Tumors contain a small population of cancer stem cells (CSC) proposed to be responsible for tumor maintenance and relapse. Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate CSCs in different cancer types. This study used the Aldefluor® assay and fluorescence-activated cell sorting (FACS) analysis to isolate ALDH1^high cells from five human sarcoma cell lines and one primary chordoma cell line. ALDH1^high cells range from 0.3% (MUG-Chor1) to 4.1% (SW-1353) of gated cells. Immunohistochemical staining, analysis of the clone formation efficiency, and xCELLigence microelectronic sensor technology revealed that ALDH1^high cells from all sarcoma cell lines have an increased proliferation rate compared to ALDH1^low cells. By investigating of important regulators of stem cell biology, real-time RT-PCR data showed an increased expression of c-Myc, β-catenin, and SOX-2 in the ALDH1^high population and a significant higher level of ABCG2. Statistical analysis of data demonstrated that ALDH1^high cells of SW-982 and SW-1353 showed higher resistance to commonly used chemotherapeutic agents like doxorubicin, epirubicin, and cisplatin than ALDH1^low cells. This study demonstrates that in different sarcoma cell lines, high ALDH1 activity can be used to identify a subpopulation of cells characterized by a significantly higher proliferation rate, increased colony forming, increased expression of ABC transporter genes and stemness markers compared to control cells. In addition, enhanced drug resistance was demonstrated.

Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1–6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed “LPA” throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors’ phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.

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.

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

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. 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. 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. 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.

Human soft tissue sarcomas represent a rare group of malignant tumours that frequently exhibit chemotherapeutic resistance and increased metastatic potential following unsuccessful treatment. In this study, we investigated the effects of costunolide and dehydrocostus lactone, which have been isolated from Saussurea lappa using activity-guided isolation, on three soft tissue sarcoma cell lines of various origins. The effects on cell proliferation, cell cycle distribution, apoptosis induction, and ABC transporter expression were analysed. Both compounds inhibited cell viability dose- and time-dependently. IC₅₀ values ranged from 6.2 µg/mL to 9.8 µg/mL. Cells treated with costunolide showed no changes in cell cycle, little in caspase 3/7 activity, and low levels of cleaved caspase-3 after 24 and 48 h. Dehydrocostus lactone caused a significant reduction of cells in the G1 phase and an increase of cells in the S and G2/M phase. Moreover, it led to enhanced caspase 3/7 activity, cleaved caspase-3, and cleaved PARP indicating apoptosis induction. In addition, the influence of costunolide and dehydrocostus lactone on the expression of ATP binding cassette transporters related to multidrug resistance (ABCB1/MDR1, ABCC1/MRP1, and ABCG2/BCRP1) was examined using real-time RT-PCR. The expressions of ABCB1/MDR1 and ABCG2/BCRP1 in liposarcoma and synovial sarcoma cells were significantly downregulated by dehydrocostus lactone. Our data demonstrate for the first time that dehydrocostus lactone affects cell viability, cell cycle distribution and ABC transporter expression in soft tissue sarcoma cell lines. Furthermore, it led to caspase 3/7 activity as well as caspase-3 and PARP cleavage, which are indicators of apoptosis. Therefore, this compound may be a promising lead candidate for the development of therapeutic agents against drug-resistant tumours.