Human Chondrosarcoma Cell Line Research Articles

Heterogeneity of chondrosarcomas response to irradiations with X-rays and carbon ions: A comparative study on five cell lines

ObjectivesChondrosarcomas are malignant bone tumors considered as resistant to radiotherapy. To unravel mechanisms of resistance, we compared biological responses of several chondrosarcomas to X-ray irradiations in normoxia and hypoxia. Since hadrontherapy with Carbon-ions gave interesting clinical outcomes, we also investigated this treatment in vitro. MethodsFive human chondrosarcoma cell lines were used and cultured in normoxia or hypoxia. Their sensitivities to irradiations were determined by carrying out survival curves. DNA damage was monitored by γH2AX expression. Apoptosis was assessed by cell cycle analysis and Apo2.7 expression, and by evaluating PARP cleavage. Senescence was evaluated using SA β-galactosidase assay. Necrosis, and autophagy, were evaluated by RIP1 and beclin-1 expression, respectively. Mutations in relevant biological pathways were screened by whole-exome sequencing. ResultsX-ray radiations induced death in some chondrosarcomas by both apoptosis and senescence (CH2879), or by either of them (SW1353 and JJ012), whereas no death was observed in other cell lines (FS090 and 105KC). Molecularly, p21 was overexpressed when senescence was elicited. Genetic analysis allowed to identify putative genes (such as TBX3, CDK2A, HMGA2) permitting to predict cell response to irradiations. Unexpectedly, chronic hypoxia did not favor radioresistance in chondrosarcomas, and even increased the radiosensitivity of JJ012 line. Finally, we show that carbon ions triggered more DNA damages and death than X-rays. ConclusionsChondrosarcomas have different response to irradiation, possibly due to their strong genetic heterogeneity. p21 expression is suggested as predictive of X-ray-induced senescence. Surprisingly, hypoxia does not increase the radioresistance of chondrosarcomas, but as expected Carbon ion beams are more effective that X-rays in normoxia, whereas their efficiency was also variable depending on cell lines.

The gene expression and protein profiles of ADAMTS and TIMP in human chondrosarcoma cell lines induced by insulin: The potential mechanisms for skeletal and articular abnormalities in diabetes

Background:The delay in wound healing, decrease in the long bones resilience to fracture, and delay in fracture healing are among common complications DM patients, and they still remain as challenging issues to be solved. The mechanism has not been fully understood yet, but high sugar and/or insulin deficiency or unresponsiveness to insulin in blood are potential causes to blame. Extracellular matrix degradation/remodeling is one of the important mechanisms whereby cell differentiation, bone remodeling and wound repair can be regulated. ADAMTS proteins play important roles in cartilage/bone metabolism. This study aimed to determine whether ADAMTS/TIMP proteins were affected by insulin application in OUMS-27 cells.Material and methods:OUMS-27 cells were induced by 10μg/mL insulin for 1,3,7,and 11days. Cells were harvested, mRNA and protein extractions were performed. Total mRNA and cDNA levels were measured by qRT-PCR and protein levels were detected by WB.Results:ADAMTS1,5, and 7 levels were significantly decreased, while TIMP-3 levels were detected increased (mRNA/protein concentrations).Conclusions:Pathologies and disturbances of cartilage/bone metabolism, delayed fracture healing in particular, in patients with DM may result from insulin deficiency. ADAMTS genes that play a role in healing process are increased during insulin deficiency, which consequently interrupts healing process by causing cartilage ECM degradation.

Zinc protects chondrocytes from monosodium iodoacetate-induced damage by enhancing ATP and mitophagy

Osteoarthritis (OA) is characterized with articular cartilage degradation, and monosodium iodoacetate (MIA)-treated chondrocyte is the most commonly used model for mimicking OA progression. Zinc protects chondrocytes from MIA-induced damage. Here, we explored the protective effects of 25 μM zinc on 5 μM MIA-treated SW1353 cells (human chondrosarcoma cell line) through the analysis of energy metabolism- and autophagy-related parameters. We found that the exposure of SW1353 cells to MIA decreased ATP levels, expression of glycolysis-related proteins, including glucose transporter 1, hexokinase 2, and pyruvate dehydrogenase E1 component subunit alpha, and the levels of mitochondrial complex I, II, IV, and V subunits of the oxidative phosphorylation pathway. MIA treatment also decreased the expression of autophagy-related proteins, including autophagic elongation protein 5 (ATG5), ATG7, and microtubule-associated protein 1A/1B light chain 3B (LC3-II) and mitophagy-related proteins, including phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), ubiquitin, and p62. These results indicate that MIA interferes with energy metabolism and the autophagic clearance of dysfunctional mitochondria (so called mitophagy). Interestingly, zinc exposure could almost completely reverse the effects of MIA, suggesting its potential protective role against OA progression.

Abstract 864: Mutant IDH1 is essential for chondrosarcoma growth

Abstract Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including chondrosarcomas. IDH mutations detected in human cancers invariably are heterozygous missense substitutions. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into 2-hydroxyglutarate (D-2HG), an oncometabolite. Due to the structural similarity between D-2HG and α-KG, it has been reported that high levels of D-2HG competitively inhibit α-KG-dependent dioxygenases such as TET, JHDM and PHD enzymes, thus contributing to tumorigenesis. We sought to determine the role of IDH1 mutations in the tumorigenesis of human chondrosarcomas by inactivating mutant IDH1 using pharmacological and genetic approaches. In our study, we employed two human chondrosarcoma cell lines, JJ012 and HT1080, that carry endogenous IDH1 mutations. IDH mutation analysis was performed by PCR-based DNA sequencing, and D-2HG levels were measured by tandem mass spectrometry. Mutant IDH1 was knocked down via siRNA and knocked out via CRISPR/Cas9. We analyzed the effect of mutant IDH1 on chondrosarcoma growth in murine xenograft models. We found that knockdown of mutant IDH1 via siRNA significantly reduced D-2HG production in chondrosarcoma cells. In addition, mutant IDH1 knockdown dramatically inhibited colony formation in the sarcoma cells. Consistently, genetic knockout of mutant IDH1 almost completely depleted D-2HG production and significantly inhibited colony formation in the chondrosarcoma cells. To assess the significance of these results in vivo, we implanted the mutant IDH1- knockout cells in nude mice and studied their capacity for tumor initiation and growth. In these models, we observed that loss of mutant IDH1 led to a marked attenuation of chondrosarcoma formation. Our findings clearly demonstrate that mutant IDH1 plays a vital role in chondrosarcoma tumor formation. By investigating the role of IDH mutations in the pathogenesis of chondrosarcomas, we aim to uncover the potential therapeutic targets against this aggressive cancer. Citation Format: Luyuan Li, Xiaoyu Hu, Josiane E. Eid, Joanna DeSalvo, Jonathan C. Trent. Mutant IDH1 is essential for chondrosarcoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 864.

Abstract 156: Visfatin induces LOX expression and promotes tumor metastasis through the Src/Akt signaling pathway in human chondrosarcoma cells

Abstract Chondrosarcoma, a common primary bone malignancy that affects the joints and bones, is associated with metastases in the lung, especially in cases of high-grade chondrosarcoma. The protein lysyl oxidase (LOX) can contribute to tumor growth and metastasis, while the adipokine visfatin is pro-angiogenic and facilitates tumorigenesis as well as metastasis. However, it is unclear as to whether any association exists between visfatin and LOX expression in human chondrosarcoma metastasis. We have discovered that visfatin induces the migration, invasion and increased expression of LOX mRNA and protein in human chondrosarcoma cell lines (JJ012 and SW1353). Pretreatment of these cell lines with Src and Akt inhibitors, or transfecting them with small interfering RNAs (siRNAs), inhibited visfatin-induced chondrosarcoma metastasis and LOX expression. Thus, visfatin promotes chondrosarcoma metastasis by inducing LOX expression via the Src/Akt signaling pathway in the JJ012 cell line. microRNA analysis revealed that visfatin-induced Src/Akt signaling is responsible for downregulating mature miR-26b-5p, which targets the 3' untranslated region (3`UTR) of LOX but does not affect primary and precursor miR-26b-5p expression. This downregulation of miR-26b-5p expression by visfatin was dose-dependent. Pretreatment with Src and Akt inhibitors or transfection with siRNA reversed miR-26b-5p expression. The migration and invasion ability of JJ012 cells was reduced when they were transfected with the miR-26b-5p mimic. In summary, our data demonstrate that visfatin induces LOX expression and promotes chondrosarcoma cell migration and invasion through the Src/Akt signaling pathway, by inhibiting miR-26b-5p. Citation Format: Cheng-Chieh Yu, Chih-Yang Lin, Chih-Hsin Tang. Visfatin induces LOX expression and promotes tumor metastasis through the Src/Akt signaling pathway in human chondrosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 156.

VE-822 mediated inhibition of ATR signaling sensitizes chondrosarcoma to cisplatin via reversion of the DNA damage response.

IntroductionCisplatin has been reported to elicit the DNA damage response (DDR) via activation of the ATR-Chk1 pathway, which in turn contributes to the induction of cisplatin resistance. Inhibition of ATR-Chk1 signaling reverses cisplatin resistance in some cancers. However, the influence of inhibiting ATR-Chk1 signaling on cisplatin resistance in chondrosarcoma cancer has not been reported.Materials and methodsWe compared the expression levels of ATR kinases in human nasopharyngeal carcinoma, choriocarcinoma and chondrosarcoma cell lines. We inhibited ATR kinase function with VE-822, a selective ATR inhibitor, and suppressed ATR kinase expression with shRNA. Western blotting, the CCK-8 assay, cell cycle distribution assay and apoptosis analysis were used to study the influence of inhibiting ATR-Chk1 signaling on reversing cisplatin resistance in chondrosarcoma cell lines.ResultsWe found that chondrosarcoma cells expressed very low basal levels of phosphorylated ATR, but cisplatin treatment induced the activation of ATR-Chk1 signaling in a dose- and time-dependent manner, suggesting the induction of DDR. As expected, ATR inhibition with VE-822 reversed cisplatin-induced DDR and enhanced cisplatin-induced activation of H2AX, which is an important marker of DNA damage. Meanwhile, ATR inhibition by RNA interference also reversed DDR and promoted DNA damage. Furthermore, both pharmacological and molecular inhibition of ATR accelerated cisplatin-induced inhibition of cell proliferation and cell death.ConclusionOur results suggested that inhibiting ATR activation promoted cisplatin-induced cell death via reversion of DDR, and VE-822 may be a valuable strategy for the prevention of cisplatin resistance in chondrosarcoma.

Anti-Apoptotic Effects of Docosahexaenoic Acid in IL-1β-Induced Human Chondrosarcoma Cell Death through Involvement of the MAPK Signaling Pathway

Osteoarthritis (OA) is a degenerative disease characterized by progressive articular cartilage destruction and joint marginal osteophyte formation with different degrees of synovitis. Docosahexaenoic acid (DHA) is an unsaturated fatty acid with anti-inflammatory, antioxidant, and antiapoptotic functions. In this study, the human chondrosarcoma cell line SW1353 was cultured in vitro, and an OA cell model was constructed with inflammatory factor IL-1β stimulation. After cells were treated with DHA, cell apoptosis was measured. Western blot assay was used to detect protein expression of apoptosis-related factors (Bax, Bcl-2, and cleaved caspase-3) and mitogen-activated protein kinase (MAPK) signaling pathway family members, including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38 MAPK. Our results show that IL-1β promotes the apoptosis of SW1353 cells, increases the expression of Bax and cleaved caspase-3, and activates the MAPK signaling pathway. In contrast, DHA inhibits the expression of IL-1β, inhibits IL-1β-induced cell apoptosis, and has a certain inhibitory effect on the activation of the MAPK signaling pathway. When the MAPK signaling pathway is inhibited by its inhibitors, the effects of DHA on SW1353 cells are weakened. Thus, DHA enhances the apoptosis of SW1353 cells through the MAPK signaling pathway.

Depletion of mutant IDH1 impairs chondrosarcoma growth by downregulating integrins.

11031 Background: chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including chondrosarcomas. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into 2-hydroxyglutarate (D-2HG), an oncometabolite. We sought to determine the role of IDH1 mutation in the tumorigenesis of human chondrosarcomas by inactivating mutant IDH1 (mIDH1) using genetic approaches. Methods: we employed two human chondrosarcoma cell lines, JJ012 and HT1080 that carry endogenous IDH1 mutations. D-2HG levels were measured by tandem mass spectrometry. mIDH1 was knocked out via the CRISPR/Cas9 technique. Colony-forming ability and cell migration were evaluated using soft agar assay and Transwell assay, respectively. The effect of mIDH1 on chondrosarcoma growth was analyzed in murine xenograft models. RNA-seq from total RNA was performed to identify differentially expressed genes (DEGs) between the parental and knockout cells. The biological functions of DEGs were identified through Ingenuity pathway analysis. Results: we found that mIDH1 knockout almost completely depleted D-2HG production, dramatically inhibited colony formation by 35% - 60% in JJ012 ( p < 0.01) and by 40% - 80% in HT1080 ( p < 0.01), and significantly reduced the migratory cell numbers by around 40% in both cell lines ( p < 0.01). Moreover, we observed that loss of mIDH1 led to a marked attenuation of chondrosarcoma formation, whereby the mean volume of the tumors derived from mIDH1-knockout cells was approximately 30% of that of control tumors, in both cell lines (JJ012 and HT1080) ( p < 0.01). Interestingly, RNA-seq data showed a significant downregulation of integrin genes such as ITGA10, ITGA5 and ITGB5 in response to mIDH1 knockout (adjusted p < 0.001). These results were confirmed by qRT-PCR and Immunoblotting. Conclusions: our findings demonstrate that mutant IDH1 plays a vital role in chondrosarcoma tumor formation through aberrant activation of integrins.

Therapeutic effect of palbociclib in chondrosarcoma: implication of cyclin-dependent kinase 4 as a potential target

BackgroundChondrosarcoma is a malignant cartilaginous neoplasm of the bone which resistant to radiation therapy and chemotherapy. Cyclin-dependent kinase 4 (CKD4) is highly expressed in human cancer, and palbociclib, the inhibitor of CDK4 has been used clinically under FDA approval for application in cancer therapeutic remedies. However, the level of CDK4 and the treatment possibility in chondrosarcoma require further exploration. Thus, we aim to investigate the level of CDK4 and accompanying therapeutic effects of palbociclib in chondrosarcoma.MethodsWe used immunohistochemistric analysis to evaluate human CDK4 productions in chondrosarcoma tissues. The inhibitory expression of CDK4 by siRNA or palbociclib on cell proliferation, invasion, migration, apoptosis and cycle arrest of chondrosarcoma were determined by MTT, wound healing, transwell and flow cytometry. CDK4/Rb signaling pathway were determined by western blot and Immunofluorescence assay. The inhibition effect of palbociclib on tumor growth within the bone were determined by bioluminescence imaging in vivo.ResultsCDK4 was found to express significantly in human chondrosarcoma samples. The enhanced levels of CDK4 were interlinked with malignant metastasis and undesirable prognosis of chondrosarcoma patients. CDK4 was also highly expressed in human chondrosarcoma cell lines and its inhibition by specific siRNA and palbociclib lead to a decrease in cell proliferation, accompanied by the phosphorylation of Rb. Furthermore, palbociclib also induced cell cycle arrest in G1 phase and decreased cell migration and invasion via CDK4/Rb signaling pathway. Administration of palbociclib in vivo could reduce tumor burden in chondrosarcoma.ConclusionsIn summary, these data highlight CDK4 inhibitors, such as palbociclib, as potential promising therapeutics in the treatment of human chondrosarcoma.

Recruitment of Brd3 and Brd4 to acetylated chromatin is essential for proinflammatory cytokine-induced matrix-degrading enzyme expression

BackgroundProinflammatory cytokines, which can upregulate the expression of matrix-degrading enzymes in chondrocytes, play important roles in the development of osteoarthritis. BET family proteins, acting as the “readers” of acetylated modifications on histones, have been linked to transcriptional regulation. And a BET protein inhibitor, I-BET151, has been shown to inhibit the induction of matrix-degrading enzymes by proinflammatory cytokines in chondrocytes. Our objective is to clarify the role and mechanism of BET proteins on matrix-degrading enzyme gene expression by using a human chondrosarcoma cell line (SW1353).MethodsWe pretreated SW1353 cells with I-BET151 prior to treatment with IL-1β or TNF-α and then checked the expression of four matrix-degrading enzyme genes (MMP1, MMP3, MMP13, and ADAMTS4). We performed knockdown of BET protein family members (BRD2, BRD3, and BRD4) with corresponding siRNAs in SW1353 cells prior to treatment with IL-1β or TNF-α and checked the expression of the matrix-degrading enzyme genes. We evaluated Brd-mediated transcriptional regulation on the matrix-degrading enzyme genes by ChIP assay.ResultsWe confirmed that I-BET151 could suppress the IL-1β- or TNF-α-induced expression of MMP1, MMP3, MMP13, and ADAMTS4 in SW1353 cells. Brd3 and Brd4 were required for the IL-1β- or TNF-α-induced expression of matrix-degrading enzyme genes in SW1353 cells. We revealed that inducible acetylation of H4k5/8/12 and the recruitment of Brd3, Brd4, and p-TEFb to chromatin were involved in IL-1β- or TNF-α-induced transcription.ConclusionsOur findings suggested that Brd3 and Brd4 were essential for the IL-1β- or TNF-α-induced transcription of matrix-degrading enzyme genes, and recruitment of Brd3 and Brd4 to chromatin of these genes played the main role in this process.

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.

MLN4924, a Protein Neddylation Inhibitor, Suppresses the Growth of Human Chondrosarcoma through Inhibiting Cell Proliferation and Inducing Endoplasmic Reticulum Stress-Related Apoptosis.

Chondrosarcoma, a heterogeneous malignant bone tumor, commonly produces cartilage matrix, which generally has no response to conventional therapies. Studies have reported that MLN4924, a NEDD8-activating enzyme inhibitor, achieves antitumor effects against numerous malignancies. In this study, the suppressive effects of MLN4924 on human chondrosarcoma cell lines were investigated using in vitro and in vivo assays, which involved measuring cell viability, cytotoxicity, apoptosis, proliferation, cell cycles, molecule-associated cell cycles, apoptosis, endoplasmic reticulum (ER) stress, and tumor growth in a xenograft mouse model. Our results demonstrated that MLN4924 significantly suppressed cell viability, exhibited cytotoxicity, and stimulated apoptosis through the activation of caspase-3 and caspase-7 in chondrosarcoma cell lines. Furthermore, MLN4924 significantly inhibited cell proliferation by diminishing the phosphorylation of histone H3 to cause G2/M cell cycle arrest. In addition, MLN4924 activated ER stress–related apoptosis by upregulating the phosphorylation of c-Jun N-terminal kinase (JNK), enhancing the expression of GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP, an inducer of endoplasmic ER stress–related apoptosis) and activating the cleavage of caspase-4. Moreover, MLN4924 considerably inhibited the growth of chondrosarcoma tumors in a xenograft mouse model. Finally, MLN4924-mediated antichondrosarcoma properties can be accompanied by the stimulation of ER stress–related apoptosis, implying that targeting neddylation by MLN4924 is a novel therapeutic strategy for treating chondrosarcoma.

The specific RIP1 inhibitor necrostatin-1 ameliorated degradation of ECM in human SW1353 cells

Abnormal destruction of the components of the articular extracellular matrix (ECM) such as type II collagen and aggrecan caused by advanced glycation end products (AGEs) has been considered as one of the pathological characteristics of osteoarthritis (OA). Receptor-interacting protein 1 (RIP1), an important serine/threonine kinase, possesses a variety of biological functions including cell proliferation, survival and death. The physiological roles of RIP1 in OA have not been reported before. Here, we found that AGEs increased the expression of RIP1 in human chondrosarcoma cell line SW1353 cells. Importantly, we found that antagonism of RIP1 using its specific inhibitor necrostatin-1 (Nec-1) ameliorated AGE-induced degradation of type II collagen and aggrecan in SW1353 cells. We also found that treatment with Nec-1 reduced the expression of MMP-3 and MMP-13 but restored the expression of Tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Also, our results indicate that Nec-1 inhibited AGE-induced expression of ADAMTS-4 and ADAMTS-5. Mechanistically, we found that Nec-1 treatment inhibited the activation of JNK and the transcriptional factor AP-1 by reducing the expressions of c-Fos and c-Jun, the two main components of AP-1. Additionally, we found that Nec-1 treatment abolished AGE-induced activation of the transcriptional factor NF-κB by suppressing the nuclear translocation of p65. These findings suggest that RIP1 might be an important therapeutic target of OA.

Inhibition of the Expression of Matrix Metalloproteinases in Articular Chondrocytes by Resveratrol through Affecting Nuclear Factor-Kappa B Signaling Pathway.

In the present study, we tried to examine whether resveratrol regulates the expression of matrix metalloproteinases (MMPs) through affecting nuclear factor-kappa B (NF-κB) in articular chondrocytes. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of resveratrol on IL-1β-induced secretion of MMP-3 was investigated in rabbit articular chondrocytes using western blot analysis. To elucidate the action mechanism of resveratrol, effect of resveratrol on IL-1β-induced NF-κB signaling pathway was investigated in SW1353, a human chondrosarcoma cell line, by western blot analysis. The results were as follows: (1) resveratrol inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) resveratrol reduced the secretion of MMP-3; (3) resveratrol inhibited IL-1β-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), and thus phosphorylation and degradation of inhibitory kappa Bα (IκBα); (4) resveratrol inhibited IL-1β-induced phosphorylation and nuclear translocation of NF-κB p65. This, in turn, led to the down-regulation of gene expression of MMPs in SW1353 cells. These results suggest that resveratrol can regulate the expression of MMPs through affecting NF-κB by directly acting on articular chondrocytes.

Structure-activity relationship study of hypoxia-activated prodrugs for proteoglycan-targeted chemotherapy in chondrosarcoma

Due to an abundant chondrogenic, poorly vascularized and particularly hypoxic extracellular matrix, chondrosarcoma, a malignant cartilaginous tumour, is chemo- and radio-resistant. Surgical resection with wide margins remains the mainstay of treatment. To address the lack of therapy, our strategy aims to increase anticancer drugs targeting and delivery in the tumour, by leveraging specific chondrosarcoma hallmarks: an extensive cartilaginous extracellular matrix, namely the high negative fixed charge density and severe chronic hypoxia. A dual targeted therapy for chondrosarcoma was investigated by conjugation of a hypoxia-activated prodrug (HAP) to quaternary ammonium (QA) functions which exhibit a high affinity for polyanionic sites of proteoglycans (PGs), the major components of the chondrosarcoma extracellular matrix. Based on preclinical results, an imidazole prodrug, ICF05016, was identified and provided the basis for a lead optimization study. A series of 27 QA-phosphoramide mustard conjugates, differing by the type of QA function and the length of the alkyl linker, was yielded by a common multi-step sequence involving phosphorylation of a key 2-nitroimidazole alcohol. Then, a screening was realized by surface plasmon resonance technology to assess biomolecular interactions between QA derivatives and aggrecan, the most abundant PG in chondrosarcoma. Results revealed that affinity depends more on the type of QA function, than on the linker length. Moreover, the presence of a benzyl group enhanced affinity to aggrecan. Twelve compounds were shortlisted and evaluated for antiproliferative activity (i.e., growth inhibiting concentration 50), under normoxic and hypoxic conditions using the human extraskeletal myeloid chondrosarcoma cell line (HEMC-SS). For all prodrugs, hypoxic selectivity was maintained and even increased, compared with the lead. From this study, compound 31f emerged as the most effective PG-targeted HAPs with a dissociation constant of 2.10 μM in the SPR experiment, a hypoxia cytotoxicity ratio of 24 and an efficient reductive cleavage under chemical and enzymatic conditions.

MiR‑204‑5p inhibits the occurrence and development of osteoarthritis by targeting Runx2.

One of the hallmarks of osteoarthritis (OA) development is endochondral ossification, in which Runt-related transcription factor-2 (Runx2) is aberrantly expressed. Runx2 was previously identified to be regulated by microRNA-204-5p (miR-204-5p). The aim of the present study was to investigate the potential function of miR-204-5p regulating Runx2 during the development of OA and the underlying molecular mechanism. The expression levels of miR-204-5p and Runx2 were determined in tissue specimens. Rat OA models were established by transecting the anterior and posterior cruciate ligaments and removing the meniscus. Rats were treated with miR-204-5p agomir and miR-204-5p negative control (NC). All in vitro experiments were performed using rat primary chondrocytes and the SW-1353 human bone chondrosarcoma cell line. It was identified that the expression of miR-204-5p was significantly decreased, whereas Runx2 was significantly increased, in human OA tissues compared with in non-OA tissues, and levels were inversely associated with each other in primary chondrocytes and chondrosarcoma cells. Overexpression of miR-204-5p decreased the proliferation of chondrocytes and SW-1353 cells. Using a luciferase reporter assay, Runx2 was identified to be a direct target of miR-204-5p in chondrocytes and overexpressed miR-204-5p altered the expression of collagens II, X and matrix metalloproteinase (MMP)-1 and MMP-13 in primary chondrocytes and SW-1353 cells. Histological analysis revealed that miR-204-5p treatment ameliorated the OA-like phenotype that is reflected by assessment of cartilage thickness and Mankin's score. Runx2 expression was gradually increased as the rats increased in age. At 10 weeks of miR-204-5p agomir treatment, altered expression levels of collagens II and X, cartilage oligomeric matrix protein fragment, aggrecan, MMP-1 and MMP-13 were observed in the treatment group compared with the NC group. In conclusion, the results of the present study indicated that miR-204-5p decreases chondrocyte proliferation and ameliorates the OA-like phenotype in rats with surgically induced OA by targeting Runx2.

Synergistic anti-proliferative effects of mTOR and MEK inhibitors in high-grade chondrosarcoma cell line OUMS-27

Chondrosarcoma is a malignant bone tumor that produces cartilaginous neoplastic tissue. Owing to the absence of an effective adjuvant therapy, high-grade chondrosarcoma has a poor prognosis. Therefore, it is important to develop an effective adjuvant therapy to prevent the recurrence and metastasis. Mammalian target of rapamycin (mTOR), a central regulator of cell growth, metabolism, proliferation, and survival, is considered an important target for anticancer drug development. The mitogen activated protein kinase (MAPK) pathway is another highly implicated cellular pathway in cancer and is thought to have compensatory effects in response to the inhibition of the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling pathway. We investigated the mechanism of anti-proliferative effect of the mTOR inhibitor rapamycin and MAPK/ERK (MEK) inhibitor PD 0325901, and the combined effect of rapamycin and PD 0325901 on human chondrosarcoma cell line (OUMS-27). Combination therapy with rapamycin and PD 0325901 showed a stronger anti-proliferative effect on OUMS-27 cells than rapamycin monotherapy. We confirmed that the dual inhibition of the PI3K/Akt/mTOR and RAF/MEK/ERK signaling pathways had synergistic anti-proliferative effects in OUMS-27. Our results suggest that combination therapy of mTOR and MEK inhibitor could be an effective therapeutic approach against chondrosarcoma.

Interferon regulatory factor 5 (IRF5) regulates the expression of matrix metalloproteinase-3 (MMP-3) in human chondrocytes

Matrix metalloproteinase-3 (MMP-3) plays a pivotal role in the destruction of articular cartilage in osteoarthritis (OA). The regulation of gene expression of MMP-3 is complicated. Interferon regulatory factor 5 (IRF5) is a member of the interferon regulatory factor family of transcription factors. Little information regarding the biological function of IRF5 on chondrocytes and the pathogenesis of OA has been reported. In the current study, for the first time, we report that IRF5 is expressed in human primary chondrocytes and human chondrosarcoma cell line SW1353 cells. In addition, IRF5 is upregulated in response to TNF-α treatment in a dose dependent manner. Interestingly, IRF5 is significantly higher in chondrocytes from OA patients compared to those from normal subjects. Notably, IRF5 mediates TNF-α- induced expression of MMP-3 in chondrocytes. Overexpression of IRF5 promotes the expression of MMP-3, however, knockdown of IRF5 reduces the expression of MMP-3. Mechanistically, IRF5 is able to enhance the transcription of MMP-3 by binding to its promoter. Also, we found that NF-κB was involved in the effects of IRF-5 on MMP-3 expression. These findings suggest that IRF5 might be a novel pharmacological target for the treatment of OA and RA.

Analysis of IL6-protein complexes in chondrosarcoma.

Cytokines produced in the tumour microenvironment serve important roles in cancer pathogenesis or in the supression of disease progression. Metastatic chondrosarcoma is a cancer of the cartilage, and our group previously reported from a human ELISA assay that interleukin 6 (IL6) expression in JJ012 chondrosarcoma cells was 86-fold lower than that in C28 chondrocytes, indicating its role as an anti-inflammatory and anti-tumorigenic factor. Additionally, to the best of our knowledge, the study was the first to demonstrate downregulation of IL6 in a human chondrosarcoma cell line. To fully elucidate the effect of this IL6 downregulation, it is important to identify protein complexes and components that bind IL6 and potentially affect its gene expression directly or indirectly. To investigate IL6-protein interactions leading to these differences in IL6 expression, the current study performed a gel retardation electrophoretic mobility shift assay (EMSA), followed by 2D gel phoresis, in-gel trypsin digestion and proteomic mass spectral analysis. The results indicated a presence of ubiquitination enzymes in C28 chondrocytes, while none were identified in JJ012 chondrosarcoma cells. While it seems counterintuitive, it may be that the absence of ubiquitination of certain factors leads to the downregulation of IL6 expression in human chondrosarcoma. Therefore, dysregulated ubiquitination may be among the possible mechanisms for the markedly reduced IL6 expression in chondrosarcoma.

Roxatidine Attenuates Degradation of Extracellular Matrix

Degradation of extracellular matrix such as type II collagen and aggrecan induced by proinflammatory cytokines has been considered as an important hallmark of Osteoarthritis (OA). Roxatidine is a licensed specific competitive H (2) −receptor antagonist used for the treatment of gastric and duodenal ulcers. The pharmacological function of roxatidine on Osteoarthritis (OA) remains unknown. In the current study, we report that roxatidine attenuated TNF-α- induced degradation of type II collagen by suppressing the expression of MMP-3 and MMP-13 in human chondrosarcoma cell line SW1353 cells. In addition, roxatidine ameliorated TNF-α- induced reduction of aggrecan by inhibiting the expression of ADAMTS-4 and ADAMTS-5. Notably, results indicate that roxatidine ameliorated TNF-α- induced the phosphorylations of IKK, IκBα, and NF-κB p65 as well as nuclear translocation of NF-κB p65 and the transcriptional activity of NF-κB, suggesting that roxatidine abolished the activation of NF-κB signaling pathway. Our findings implicate that roxatidine might be considered as an anti-osteoarthritic agent.