Results of an Inpatient Preventive Health Care Program to Improve Quality of Life, Psychosocial Health, and Work Ability in Austria.

Objective: The Austrian Federal Pension Insurance (PVA) developed a preventive inpatient health program, "Gesundheitsvorsorge-Aktiv (GVA)," for patients with musculoskeletal disorders. Individualized modular interventions and therapeutical measures (movement optimization, movement motivation, and mental health) are designed to improve occupational participation by influencing lifestyle factors and health-related quality of life. The study aimed to evaluate the new prevention-oriented and more personalized inpatient health program GVA. Methods: Patients underwent a standard inpatient health program, with emphasis on exercise management, exercise motivation, or psychological aspects. Submodule-dependent outcomes were assessed in patients (n = 330) at the start, end of treatment, and 6 months thereafter. Quality of Life (EQ-5D-5L), psychosocial aspects of the Patient Health Questionnaire (PHQ-D), and Work Ability Index (WAI) were queried. Results: The results consistently showed positive short and long-term effects. The subjective assessments of current work ability improved while the impairment of work performance was reduced. Positive changes in the psychosocial sphere were observed, alongside improvements in the health-related quality of life. Patients in the exercise optimization module performed better in all respects. Conclusion: In summary, GVA represents a valuable preventive health measure that leads to a holistic increase in well-being and can also ensure the maintenance of the ability to work.

Activation of the Mechanosensitive Ion Channels Piezo1 and TRPV4 in Primary Human Healthy and Osteoarthritic Chondrocytes Exhibits Ion Channel Crosstalk and Modulates Gene Expression.

Osteoarthritis (OA) is the most common degenerative joint disease causing pain and functional limitations. Physical activity as a clinically relevant, effective intervention alleviates pain and promotes joint function. In chondrocytes, perception and transmission of mechanical signals are controlled by mechanosensitive ion channels, whose dysfunction in OA chondrocytes is leading to disease progression. Signaling of mechanosensitive ion channels Piezo/TRPV4 was analyzed by Yoda1/GSK1016790A application and calcium-imaging of Fura-2-loaded chondrocytes. Expression analysis was determined by qPCR and immunofluorescence in healthy vs. OA chondrocytes. Chondrocytes were mechanically stimulated using the Flexcell™ technique. Yoda1 and GSK1016790A caused an increase in intracellular calcium [Ca2+]i for Yoda1, depending on extracellularly available Ca2+. When used concomitantly, the agonist applied first inhibited the effect of subsequent agonist application, indicating mutual interference between Piezo/TRPV4. Yoda1 increased the expression of metalloproteinases, bone-morphogenic protein, and interleukins in healthy and OA chondrocytes to a different extent. Flexcell™-induced changes in the expression of MMPs and ILs differed from changes induced by Yoda1. We conclude that Piezo1/TRPV4 communicate with each other, an interference that may be impaired in OA chondrocytes. It is important to consider that mechanical stimulation may have different effects on OA depending on its intensity.

The Patient's Physiological Status at the Start Determines the Success of the Inpatient Cardiovascular Rehabilitation Program.

Multidisciplinary inpatient rehabilitation plays an important role in the recovery of patients with cardiovascular diseases (CVDs). Lifestyle changes, achieved by exercise, diet, weight loss and patient education programs, are the first steps to a healthier life. Advanced glycation end products (AGEs) and their receptor (RAGE) are known to be involved in CVDs. Clarification on whether initial AGE levels can influence the rehabilitation outcome is important. Serum samples were collected at the beginning and end of the inpatient rehabilitation stay and analyzed for parameters: lipid metabolism, glucose status, oxidative stress, inflammation and AGE/RAGE-axis. As result, a 5% increase in the soluble isoform RAGE (sRAGE) (T0: 891.82 ± 44.97 pg/mL, T1: 937.17 ± 43.29 pg/mL) accompanied by a 7% decrease in AGEs (T0: 10.93 ± 0.65 µg/mL, T1: 10.21 ± 0.61 µg/mL) was shown. Depending on the initial AGE level, a significant reduction of 12.2% of the AGE activity (quotient AGE/sRAGE) was observed. We found that almost all measured factors improved. Summarizing, CVD-specific multidisciplinary rehabilitation positively influences disease-associated parameters, and thus provides an optimal starting point for subsequent disease-modifying lifestyle changes. Considering our observations, the initial physiological situations of patients at the beginning of their rehabilitation stay seem to play a decisive role regarding the assessment of rehabilitation success.

Shikonin derivatives cause apoptosis and cell cycle arrest in human chondrosarcoma cells via death receptors and MAPK regulation.

BACKGROUND: Although chondrosarcoma is the second most common primary malignant bone tumor, treatment options are limited due to its extensive resistance to a chemo- and radiation therapy. Since shikonin has shown potent anticancer activity in various types of cancer cells, it represents a promising compound for the development of a new therapeutic approach. METHODS: The dose-relationships of shikonin and its derivatives acetylshikonin and cyclopropylshikonin on two human chondrosarcoma cell lines were measured using the CellTiter-Glo®. The changes in the cell cycle were presented by flow cytometry. Protein phosphorylation and expression apoptotic markers, MAPKs and their downstream targets were analyzed using western blotting and gene expression were evaluated using RT-qPCR. RESULTS: Chondrosarcoma cells showed a dose-dependent inhibition of cell viability after treatment with shikonin and its derivatives, with the strongest effect for shikonin and IC50 values of 1.3 ± 0.2 µM. Flow cytometric measurements revealed a G2/M arrest of the cells after treatment. Protein and gene expression analysis demonstrated a dose-dependent downregulation of survivin and XIAP, and an upregulation of Noxa, γH2AX, cleaved caspase-8, -9, -3, and -PARP. Furthermore, the expression of various death receptors was modulated. As MAPK signaling pathways play a key role in tumor biology, their phosphorylation pattern and their corresponding downstream gene regulation were analyzed. Treatment with shikonin derivatives caused an inhibition of pSTAT3 and an increase of pAKT and the MAPKs pERK, pJNK, and pp38 in a dose-dependent manner. CONCLUSIONS: These data demonstrated the significant anti-tumorigenic effect of shikonin derivatives in chondrosarcoma and encourage further research.

Nuclear Magnetic Resonance Treatment Accelerates the Regeneration of Dorsal Root Ganglion Neurons in vitro.

Functional recovery from peripheral nerve injuries depends on a multitude of factors. Schwann cells (SCs) are key players in the regenerative process as they develop repair-specific functions to promote axon regrowth. However, chronically denervated SCs lose their repair phenotype, which is considered as a main reason for regeneration failure. Previous studies reported a modulatory effect of low nuclear magnetic resonance therapy (NMRT) on cell proliferation and gene expression. To provide first insight into a possible effect of NMRT on cells involved in peripheral nerve regeneration, this study investigated whether NMRT is able to influence the cellular behavior of primary SC and dorsal root ganglion (DRG) neuron cultures in vitro. The effect of NMRT on rat SCs was evaluated by comparing the morphology, purity, proliferation rate, and expression levels of (repair) SC associated genes between NMRT treated and untreated SC cultures. In addition, the influence of (1) NMRT and (2) medium obtained from NMRT treated SC cultures on rat DRG neuron regeneration was examined by analyzing neurite outgrowth and the neuronal differentiation status. Our results showed that NMRT stimulated the proliferation of SCs without changing their morphology, purity, or expression of (repair) SC associated markers. Furthermore, NMRT promoted DRG neuron regeneration shown by an increased cell survival, enhanced neurite network formation, and progressed neuronal differentiation status. Furthermore, the medium of NMRT treated SC cultures was sufficient to support DRG neuron survival and neurite outgrowth. These findings demonstrate a beneficial impact of NMRT on DRG neuron survival and neurite formation, which is primarily mediated via SC stimulation. Our data suggest that NMRT could be suitable as a non-invasive auxiliary treatment option for peripheral nerve injuries and encourage future studies that investigate the effect of NMRT in a physiological context.

Shikonin Derivatives Inhibit Inflammation Processes and Modulate MAPK Signaling in Human Healthy and Osteoarthritis Chondrocytes.

Osteoarthritis (OA) is the most common joint disorder and is characterized by the degeneration of articular cartilage. To develop new therapeutic approaches, we investigated the effect of shikonin derivatives on inflammation, MMP expression, and the regulation of MAPK signaling in human healthy (HC) and OA chondrocytes (pCH-OA). Viability was analyzed using the CellTiter-Glo® Assay. Inflammatory processes were investigated using a proteome profiler™ assay. Furthermore, we analyzed the effects of the shikonin derivatives by protein expression analysis of the phosphorylation pattern and the corresponding downstream gene regulation using RT-qPCR. Both HC and pCH-OA showed a dose-dependent decrease in viability after treatment. The strongest effects were found for shikonin with IC50 values of 1.2 ± 0.1 µM. Shikonin counteracts the inflammatory response by massively reducing the expression of the pro-inflammatory mediators. The phosphorylation level of ERK changed slightly. pJNK and pp38 showed a significant increase, and the downstream targets c/EBPs and MEF2c may play a role in the cartilage homeostasis. STAT3 phosphorylation decreased significantly and has a chondroprotective function through the regulation of cyclin D1 and Sox9. Our results demonstrate for the first time that shikonin derivatives have extensive effects on the inflammatory processes, MAPKs, and IL6/STAT3 downstream regulation in healthy and OA chondrocytes.

The Association of Blood Biomarkers and Body Mass Index in Knee Osteoarthritis: A Cross-Sectional Study.

OBJECTIVE: Despite massive efforts, there are no diagnostic blood biomarkers for knee osteoarthritis (KOA). This study investigated several candidate diagnostic biomarkers and the metabolic phenotype in end-stage KOA in the context of obesity. DESIGN: In this cross-sectional study, adult patients undergoing knee arthroplasty were enrolled and KOA severity was assessed using the Lequesne index. Blood biomarkers with an important role in obesity, the metabolic syndrome, or KOA (oxidized form of low-density lipoprotein [oxLDL], advanced glycation end product [AGE], soluble AGE receptor [sRAGE], fatty acid binding protein 4 [FABP4], phospholipase A2 group IIA [PLA2G2A], fibroblast growth factor 23 [FGF-23], ghrelin, leptin, and resistin) were measured using enzyme-linked immunosorbent assay (ELISA; n = 70) or Luminex technique (subgroup of n = 35). H1-NMR spectroscopy was used for the quantification of metabolite levels (subgroup of n = 31). The hip-knee-ankle angle was assessed. Multivariable and multivariate regression analysis was used to examine the relationship of biomarkers with body mass index (BMI) and KOA severity in complete case and multiple imputation analysis. RESULTS: While most of the investigated biomarkers were not associated with KOA severity, FABP4 and leptin were found to correlate with BMI and gender. Resistin was associated with Lequesne index in complete case analysis. Using a targeted metabolomics approach, BMI-dependent changes in the metabolome were hardly visible. CONCLUSIONS: Our findings confirm studies on FABP4, leptin, and resistin with regard to obesity and the metabolic syndrome. There was no association of the investigated biomarkers with KOA severity, most likely due to the patient selection (end-stage KOA patients). Based on this absence of BMI-dependent changes in the metabolome, we might assume that BMI is not correlated with KOA severity in this specific patient group.

Improving the measurement of oral health-related quality of life: Rasch model of the oral health impact profile-14.

OBJECTIVES: The 14-item Oral Health Impact Profile (OHIP-14) is the most frequently used instrument to measure oral health-related quality-of-life (OHRQoL) in adults. Despite its popularity, its psychometric properties have been predominantly investigated based on the classical test theory while the fundamental principles of measurement have not been fully assessed. Therefore, our aim was to investigate to what extent the OHIP-14 meets the fundamental requirments of measurements. METHODS: We used the Rasch model to explore person-item-targeting, unidimensionality, local independence of items, invariance (differential-item-functioning, DIF), and the order of thresholds between response-options in the German version OHIP-14. We used data from osteoarthritis patients because hand disabilities and joint pain might influence oral hygiene. Furthermore, osteoarthritis in the temporomandibular-joint directly affects oral functioning. RESULTS: Five-hundred sixteen patients were included (mean age 66.5 years [±10.2; ranging from 34 to 89]; 71.3% [368] females). The OHIP-14 median total score was 0 (interquartile-range from 0 to 4), indicating a right-skewed distribution because many patients reported good OHRQoL. The instrument was found unidimensional. However, there was strong evidence of local dependency, disordered thresholds between response-options, and age-related DIF for item 5. A revised scoring scheme with three instead of five answer-options in all items and eliminating two items resolving local dependency, the newly adapted OHIP-12, showed better reliability and item-fit to the Rasch model than the original OHIP-14. CONCLUSIONS: This study assesses, for the first time, the OHIP-14 in terms of fundamental principles of measurement and proposes an item-reduced OHIP-12 as a psychometrically more accurate version of the instrument. CLINICAL SIGNIFICANCE: The Rasch model is essential to ensure instruments' precision and clinical meaningfulness when measuring OHRQoL in clinical practice and research. The OHIP-12, derived from the OHIP-14 by deleting two items due to local dependency, with a revised scoring scheme for all items distinguishing three answer-options instead of five, represents a psychometrically improved version of the instrument.

Fatty Acid-Binding Protein 4 (FABP4) Is Associated with Cartilage Thickness in End-Stage Knee Osteoarthritis.

BACKGROUND: There is no single blood biomarker for the staging of knee osteoarthritis (KOA). The purpose of this study was to assess the relationship of obesity, serum biomarkers, the hip-knee-ankle angle (HKAA) with sonographic cartilage thickness. METHODS: We conducted a cross-sectional study of n = 33 patients undergoing knee arthroplasty. Body mass index (BMI) was recorded, and patients were grouped based on BMI. Serum blood samples were collected, and the following biomarkers were measured using the ELISA technique (subgroup of n = 23): oxidized low-density lipoprotein (oxLDL), soluble receptor for advanced glycation end-products (sRAGE), fatty acid-binding protein 4 (FABP4), membrane-bound phospholipase A2 (PLA2G2A). The HKAA was analyzed on full-length limb standing x-ray images. Cartilage thickness was assessed on ultrasound images. Multivariable regression analysis was performed to account for confounding. RESULTS: After adjusting for age, gender, and HKAA, obese patients had thicker medial femoral cartilage (ß = 0.165, P = 0.041). Furthermore, lateral cartilage thickness was negatively correlated with FABP4 level after adjusting for of age, gender, BMI, and HKAA (ß = -0.006, P = 0.001). Confirming previous studies, after adjustment, FABP4 level was associated with a higher BMI group (ß = 42.99, P < 0.001). None of the other markers (oxLDL, PLA2G2A, and sRAGE) was associated with BMI or cartilage thickness. DISCUSSION: Our results indicate that BMI has a weak, positive association with cartilage thickness in end-stage KOA patients. FABP4 levels were negatively associated with cartilage thickness. While our study is limited by a small sample size, these results further highlight the role of FABP4 as promising biomarkers of burden of disease in KOA.

Nuclear Magnetic Resonance Therapy Modulates the miRNA Profile in Human Primary OA Chondrocytes and Antagonizes Inflammation in Tc28/2a Cells.

Nuclear magnetic resonance therapy (NMRT) is discussed as a participant in repair processes regarding cartilage and as an influence in pain signaling. To substantiate the application of NMRT, the underlying mechanisms at the cellular level were studied. In this study microRNA (miR) was extracted from human primary healthy and osteoarthritis (OA) chondrocytes after NMR treatment and was sequenced by the Ion PI Hi-Q™ Sequencing 200 system. In addition, T/C-28a2 chondrocytes grown under hypoxic conditions were studied for IL-1ß induced changes in expression on RNA and protein level. HDAC activity an NAD(+)/NADH was measured by luminescence detection. In OA chondrocytes miR-106a, miR-27a, miR-34b, miR-365a and miR-424 were downregulated. This downregulation was reversed by NMRT. miR-365a-5p is known to directly target HDAC and NF-ĸB, and a decrease in HDAC activity by NMRT was detected. NAD+/NADH was reduced by NMR treatment in OA chondrocytes. Under hypoxic conditions NMRT changed the expression profile of HIF1, HIF2, IGF2, MMP3, MMP13, and RUNX1. We conclude that NMRT changes the miR profile and modulates the HDAC and the NAD(+)/NADH signaling in human chondrocytes. These findings underline once more that NMRT counteracts IL-1ß induced changes by reducing catabolic effects, thereby decreasing inflammatory mechanisms under OA by changing NF-ĸB signaling.

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

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 G2/M phase arrest, which was accompanied by a decrease in the number of cells at the G1 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.

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

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 (ß = -0.11, P = 0.03). Metformin slightly reduced expression in ADAMTS5 (ß = 0.34, P = 0.04), HIF-1a (ß = 0.39, P = 0.04), IL4 (ß = 0.30, P = 0.02), MMP1 (ß = 0.47, P < 0.01), and SOX9 (ß = 0.37, P = 0.03). The hip-knee-ankle angle and proton pump inhibitors (PPIs) intake were associated with reduced SOX9 expression (ß = 0.23, P < 0.01; ß = 2.39, P < 0.01). Higher C-reactive protein (CRP) levels were associated with increased MMP1 expression (ß = -0.16, P = 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.

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

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.

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

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-1ß. NMRT treated cells were tested for changes in histamine induced Ca2+ release by fura-2 calcium imaging. The effects of IL-1ß 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-1ß 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-1ß-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.

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

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.

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

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.

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

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 p38ß 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.

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

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.

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

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.

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

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.