Establishment and Characterization of a Novel Dedifferentiated Chondrosarcoma Cell Line DDCS2

Background The dedifferentiated variant of chondrosarcoma is highly aggressive and carries an especially grim prognosis. While chemotherapeutics has failed to benefit patients with dedifferentiated chondrosarcoma significantly, preclinical chemosensitivity studies have been limited by a scarcity of available cell lines. There is, therefore, an urgent need to expand the pool of available cell lines. Methods We report the establishment of a novel dedifferentiated chondrosarcoma cell line DDCS2, which we isolated from the primary tumor specimen of a 60-year-old male patient. We characterized its short tandem repeat (STR) DNA profile, growth potential, antigenic markers, chemosensitivity, and oncogenic spheroid and colony-forming capacity. Results DDCS2 showed a spindle to polygonal shape and an approximate 60-hour doubling time. STR DNA profiling revealed a unique genomic identity not matching any existing cancer cell lines within the ATCC, JCRB, or DSMZ databases. There was no detectable contamination with another cell type. Western blot and immunofluorescence assays were consistent with a mesenchymal origin, and our MTT assay revealed relative resistance to conventional chemotherapeutics, which is typical of a dedifferentiated chondrosarcoma. Under ex vivo three-dimensional (3D) culture conditions, the DDCS2 cells produced spheroid patterns similar to the well-established CS-1 and SW1353 chondrosarcoma cell lines. Conclusion Our findings confirm DDCS2 is a novel model for dedifferentiated chondrosarcoma and therefore adds to the limited pool of current cell lines urgently needed to investigate the chemoresistance within this deadly cancer.

Anti-Osteoarthritic Mechanisms of Chrysanthemum zawadskii var. latilobum in MIA-Induced Osteoarthritic Rats and Interleukin-1β-Induced SW1353 Human Chondrocytes

Background and objectives: Chrysanthemum zawadskii var. latilobum (CZ), which has traditionally been used as a oriental tea in Asia, is known to have anti-inflammatory effects in osteoarthritis (OA). But the mechanism of these effects has not been made clear and it needs to be elucidated specifically for the clinical use of CZE in OA. Materials and Methods: To reveal this mechanism, we first identified which biomarkers were expressed in the joints of rats in which OA had been induced with monosodium iodoacetate and determined whether CZ extract (CZE) could normalize these biomarkers in the progression of OA. The anti-osteoarthritis effect of CZE was evaluated for its capability to inhibit levels of extracellular matrix (ECM)-degrading enzymes and enhance ECM synthesis. We also sought to identify whether the marker compound of CZE, linarin, has anti-osteoarthritic effects in the human chondrosarcoma cell line SW1353. Results: The changes in matrix metalloproteinases (MMPs) were remarkable: among them, MMP-1, MMP-3, MMP-9 and MMP-13 were most strongly induced, whereas their expressions were inhibited by CZE dose dependently. The expressions of the ECM synthetic genes, COL2A1 and ACAN, and the transcription factor SOX9 of these genes were reduced by OA induction and significantly normalized by CZE dose dependently. SOX9 is also a repressor of ECM-degrading aggrecanases, ADAMTS-4 and ADAMTS-5, and CZE significantly reduced the levels of these enzymes dose dependently. Similar results were obtained using the human chondrosarcoma cell line SW1353 with linarin, the biologically active compound of CZE. Conclusions: These anti-osteoarthritic effects suggest that CZE has mechanisms for activating ECM synthesis with SOX9 as well as inhibiting articular ECM-degrading enzymes.

SOX9 Knockout Induces Polyploidy and Changes Sensitivity to Tumor Treatment Strategies in a Chondrosarcoma Cell Line

As most chemotherapeutic drugs are ineffective in the treatment of chondrosarcoma, we studied the expression pattern and function of SOX9, the master transcription factor for chondrogenesis, in chondrosarcoma, to understand the basic molecular principles needed for engineering new targeted therapies. Our study shows an increase in SOX9 expression in chondrosarcoma compared to normal cartilage, but a decrease when the tumors are finally defined as dedifferentiated chondrosarcoma (DDCS). In DDCS, SOX9 is almost completely absent in the non-chondroid, dedifferentiated compartments. CRISPR/Cas9-mediated knockout of SOX9 in a human chondrosarcoma cell line (HTB94) results in reduced proliferation, clonogenicity and migration, accompanied by an inability to activate MMP13. In contrast, adhesion, apoptosis and polyploidy formation are favored after SOX9 deletion, probably involving BCL2 and survivin. The siRNA-mediated SOX9 knockdown partially confirmed these results, suggesting the need for a certain SOX9 threshold for particular cancer-related events. To increase the efficacy of chondrosarcoma therapies, potential therapeutic approaches were analyzed in SOX9 knockout cells. Here, we found an increased impact of doxorubicin, but a reduced sensitivity for oncolytic virus treatment. Our observations present novel insight into the role of SOX9 in chondrosarcoma biology and could thereby help to overcome the obstacle of drug resistance and limited therapy options.

Physical Activity Prevents Cartilage Degradation: A Metabolomics Study Pinpoints the Involvement of Vitamin B6

Osteoarthritis (OA) is predominantly characterized by the progressive degradation of articular cartilage, the connective tissue produced by chondrocytes, due to an imbalance between anabolic and catabolic processes. In addition, physical activity (PA) is recognized as an important tool for counteracting OA. To evaluate PA effects on the chondrocyte lineage, we analyzed the expression of SOX9, COL2A1, and COMP in circulating progenitor cells following a half marathon (HM) performance. Therefore, we studied in-depth the involvement of metabolites affecting chondrocyte lineage, and we compared the metabolomic profile associated with PA by analyzing runners’ sera before and after HM performance. Interestingly, this study highlighted that metabolites involved in vitamin B6 salvage, such as pyridoxal 5’-phosphate and pyridoxamine 5’-phosphate, were highly modulated. To evaluate the effects of vitamin B6 in cartilage cells, we treated differentiated mesenchymal stem cells and the SW1353 chondrosarcoma cell line with vitamin B6 in the presence of IL1β, the inflammatory cytokine involved in OA. Our study describes, for the first time, the modulation of the vitamin B6 salvage pathway following PA and suggests a protective role of PA in OA through modulation of this pathway.

IL-10 Could Play a Role in the Interrelation between Diabetes Mellitus and Osteoarthritis

The association between osteoarthritis (OA), obesity and metabolic syndrome suggests an interrelation between OA and diabetes mellitus (DM). Little is known about the role of anti-inflammatory cytokine interleukin (IL)-10 in the interrelation between OA and DM. Hence, the effects of IL-10 under hyperglycemia (HG) and hyperinsulinemia (HI) in human articular chondrocytes (hAC) and chondrosarcoma cell line Okayama University Medical School (OUMS)-27 were examined. HAC and OUMS-27, cultured in normoglycemic (NG) and HG conditions were stimulated with insulin and/or IL-10. Cell survival, metabolic activity, proliferation and extracellular matrix (ECM) synthesis were immunocytochemically examined. No significant differences in vitality of hAC neither in pure NG (NGw/o) nor HG (HGw/o) conditions were found. Applying HI and/or IL-10 in both conditions reduced significantly the vitality of hAC but not of OUMS-27. HG impaired significantly hAC metabolism. When combined with HI + IL-10 or IL-10 alone it decreased also significantly hAC proliferation compared to NGw/o. In OUMS-27 it induced only a trend of impaired proliferation compared to NGw/o. hAC but not OUMS-27 reduced significantly their collagen type (col) I, SOX9 and proteoglycan (PG) synthesis in HG combined with HI +/− IL-10 compared to NGw/o. IL-10 could not moderate HI and HG effects. In contrast to hAC OUMS-27 showed limited sensitivity as DM model.