Human Chondrosarcoma Cell Line Research Articles

Inflammatory Response Modulation by Low-Dose Anti-inflammatory Drugs Treatment in an In Vitro Osteoarthritis Cellular Model.

Low-dose-medicine is based on the administration of low doses of biological regulators to restore the immunologic balance altered in the disease. Cytokines are pivotal regulators of cellular and tissue functions and impaired crosstalk, due to an imbalance between specific cytokines, it is fundamental in acute inflammation and diseases correlated to low-grade chronic inflammation. Osteoarthritis is the most prevalent arthritic disease and a leading cause of disability. In the treatment of muscle- skeletal pathologies, the therapeutic integration of conventional medicine with homotoxicology, or low-dose-medicine appears to be beneficial. This study aims to get more insights into the role of inflammatory cytokines and chemokines during the development of osteoarthritis and to evaluate a possible blocking strategy using anti-inflammatory molecules, we resort to an in vitro experimental model using an established human chondrosarcoma cell line that underwent to a well known pro-inflammatory stimulus as bacterial lipopolysaccharide. We tested the production of inflammatory-related cytokines and chemokines, and the efficacy of low-dose (LD) administration of anti-inflammatory compounds, namely IL-10 and anti-IL-1, to block inflammatory cellular pathways. Following an inflammatory insult, chondrocytes upregulated the expression of several pro-inflammatory cyto-/chemokines and this induction could be counteracted by LD IL-10 and anti-IL-1. We reported that these effects could be ascribed to an interfering effect of LD drugs with the NF-κB signaling. Our results provided a good indication that LD drugs can be effective in inhibiting the inflammatory response in chondrocytes opening the way to new therapies for the treatment of diseases such as osteoarthritis.

Preparation of Borrelia-Infected Mammalian Cells for Helium Ion Microscopy.

Preparation of mammalian cells for a Borrelia burgdorferi infection can be cumbersome especially if investigating possible cell entry processes. The initial steps of infection or entry into cells by a pathogen often involve attachment to the cell surface and plasma membrane changes. To topologically investigate with great resolution and detail these interactions of the pathogen and the mammalian cell, helium ion microscopy (HIM) can be employed. Here we describe a protocol used to define a specific multiplicity of infection (MOI) of Borrelia burgdorferi on a human chondrosarcoma cell line (SW1353) so that fine detail structures on the mammalian cell can be observed and quantified by HIM.

Apoptosis Induction Through Increased TRPV1 Activation by Synergic Effect of Melatonin and Doxorubicin in Human Osteosarcoma and Chondrosarcoma Cell Lines

Aim: We aimed to reveal the role of doxorubicin (Dox), melatonin (Mel) and transient receptor potential Vanilloid 1 (TRPV1) channels in bone and cartilage cancer cells during the treatment process. Human Bone Osteosarcoma (Saos-2/An1) and Human Chondrosarcoma (Hs 819.T) cell lines were used to prepare in-vitro experiment models. Methods: Both cell lines were cultured at 37°C. We have separated each cell line into five groups as follows: Controls, Dox, Dox+Capsazepine (Cpz), Dox+Melatonin (Mel), and combined Dox+Mel+Cpz given group. Capsaicin and capsazepine were added to cell culture mediums to activate or inactivate the TRPV1 channels, respectively. Cytosolic calcium, apoptosis, intracellular reactive oxygen, mitochondrial depolarization, caspase-3 and caspase-9 levels were measured. Results: Increased apoptotic activity was detected in doxorubicin given cell lines (Group II) when compared with the controls (p˂0.001). There was also a significantly higher apoptotic level in Dox+Mel group (Group IV), when compared with only Dox given group (p˂0.001). TRPV1 inhibition applied groups (Group III and V) have had lower apoptotic levels than other drug administered groups (p˂0.001). Conclusion: This study has indicated that apoptotic effects of Dox and Mel on both osteosarcoma and chondrosarcoma were strictly associated to TRPV1 channels, and that TRPV1 channels played an important role in whole mitochondria dependent pathways of apoptosis, which in turn may lead to increased intracellular Ca+2 levels and mitochondrial depolarization.

NADPH oxidase 4 facilitates progression of chondrosarcoma via generation of reactive oxygen species.

Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is an enzyme that regulates reactive oxygen species (ROS) generation, and its function in the development of chondrosarcoma remains unclear. In the present study, we studied NOX4 expression in chondrosarcoma by immunochemical examination, and analyzed the role of NOX4 in viability and apoptosis of human chondrosarcoma cell line SW1353 using NOX4 siRNA or NOX4 inhibitor GKT137831. NOX4 level significantly increased in tumor compared to that in para-carcinoma sample. The levels of NOX4 were positively correlated with histological grade and Musculoskeletal Tumor Society stage of the patients. NOX4 level was significantly increased in SW1353 compared with that in chondrocytes CHON-001. Knockdown of NOX4 or inhibition of NOX4 by GKT137831 both decreased generation of ROS, and induced growth inhibition and apoptosis in SW1353, accompanied with the activation of caspases (caspase-3, caspase-8 and caspses-9), upregulation of Bax, cytochrome C(cyt-c), cleaved-PARP and down-regulation of Bcl-2. Moreover, NOX4 siRNA and GKT137831 decreased the expression of p-Akt, p-ERK and p-p65 in SW1353 cells. In an in vivo study, NOX4 shRNA transfected SW1353 have shown impaired growth ability compared to the SW1353 when they were injected into the nude mice. Meanwhile, GKT137831 induced growth inhibition and apoptosis in SW1353 xenograft animals, together with increased expression of Bax, cyt-c, cleaved-PARP, and decreased expression of Bcl-2, p-Akt, p-ERK and p-p65. NOX4 plays a positive role in the development of chondrosarcoma and could serve as a promising target against chondrosarcoma clinically.

Nε-(1-Carboxymethyl)-L-lysine, an advanced glycation end product, exerts malignancy on chondrosarcoma via the activation of cancer stemness.

Despite epidemiological evidence that suggests diabetes mellitus is a risk factor for cancer, the link between diabetes mellitus and primary bone cancer is rarely discussed. Chondrosarcomas are primary malignant cartilage tumors with poor prognosis and high metastatic potential. It remains unclear whether hyperglycemia affects the stemness and malignancy of chondrosarcoma cells. Nε-(1-Carboxymethyl)-L-lysine (CML), an advanced glycation end product (AGE), is a major immunological epitope detected in the tissue proteins of diabetic patients. We hypothesized that CML could enhance cancer stemness in chondrosarcoma cells. CML enhanced tumor-sphere formation and the expression of cancer stem cell markers in human chondrosarcoma cell lines. Migration and invasion ability and the epithelial-mesenchymal transition (EMT) process were also induced by CML treatment. Moreover, CML increased the protein expression levels of the receptor for AGE (RAGE), phosphorylated NFκB-p65, and decreased the phosphorylation of AKT and GSK-3. We also found that hyperglycemia with high CML levels facilitated tumor metastasis, whereas tumor growth was not affected in the streptozotocin (STZ)-induced diabetic NOD/SCID tumor xenograft mouse models. Our results indicate that CML enhances chondrosarcoma stemness and metastasis, which may reveal the relationship between AGE and bone cancer metastasis.

Abstract 6128: Deubiquitinating enzyme Inhibitor PR-619 inducesendoplasmic reticulum stress-related apoptosisin human chondrosarcoma

Abstract It is reported that PR-619 is a deubiquitination enzyme inhibitor that can exert anti-tumor effects on a variety of malignancies. We conducted this study to explore the antitumor effects of PR-619 on chondrosarcoma in vitro and in vivo by using two different classes of human chondrosarcoma cell lines SW-11353 and JJ012. To determine these effects, we measured cell viability (MTT), apoptosis (detection of Annexin V-FITC labeling using flow cytometry) and western blot to evaluate molecules associated with apoptosis and cell stress. In addition, we used a nude mouse xenograft mouse model to determine the effect of PR-619 on tumor growth. As our data revealed, in addition to inducing BAD and activating Capase-7, Caspase-8, and PARP, PR-619 also has a dose-effect that induces cytotoxicity, proliferation restriction, and apoptosis in these human chondrosarcoma cells. PR-619 has also been found to activate endoplasmic reticulum pressure-related molecules (caspase-4 and CHOP) and other stress responses (activation of JNK and c-Jun). We conclusively demonstrated the ability of PR-619 to inhibit tumor growth while achieving minimal general toxicity in a developed xenograft mouse model of chondrosarcoma. Therefore, we conclude that PR-619 exacts an antitumor effect in conjunction with cellular and endoplasmic reticulum pressure in human chondrosarcoma. These findings suggest that PR-619 may provide a novel therapeutic strategy for chondrosarcoma. Citation Format: Kuan-Lin Kuo, Chen-Hsun Hsu, Shih-Ming Liao, Kuo-Yuan Huang. Deubiquitinating enzyme Inhibitor PR-619 inducesendoplasmic reticulum stress-related apoptosisin human chondrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6128.

The ATR Inhibitor VE-821 Enhances the Radiosensitivity and Suppresses DNA Repair Mechanisms of Human Chondrosarcoma Cells.

To overcome the resistance to radiotherapy in chondrosarcomas, the prevention of efficient DNA repair with an additional treatment was explored for particle beams as well as reference X-ray irradiation. The combined treatment with DNA repair inhibitors-with a focus on ATRi VE-821-and proton or carbon ions irradiation was investigated regarding cell viability, proliferation, cell cycle distribution, MAPK phosphorylation, and the expression of key DNA repair genes in two human chondrosarcoma cell lines. Pre-treatment with the PARPis Olaparib or Veliparib, the ATMi Ku-55933, and the ATRi VE-821 resulted in a dose-dependent reduction in viability, whereas VE-821 has the most efficient response. Quantification of γH2AX phosphorylation and protein expression of the DNA repair pathways showed a reduced regenerative capacity after irradiation. Furthermore, combined treatment with VE-821 and particle irradiation increased MAPK phosphorylation and the expression of apoptosis markers. At the gene expression and at the protein expression/phosphorylation level, we were able to demonstrate the preservation of DNA damage after combined treatment. The present data showed that the combined treatment with ATMi VE-821 increases the radiosensitivity of human chondrosarcoma cells in vitro and significantly suppresses efficient DNA repair mechanisms, thus improving the efficiency of radiotherapy.

Cytotoxic Effects of Common Irrigation Solutions on Chondrosarcoma and Giant Cell Tumors of Bone.

Irrigation is commonly used as an adjuvant treatment during the intralesional curettage of bone tumors. The goal of the present study was to analyze the in vitro cytotoxicity of commonly used irrigation solutions on chondrosarcoma and giant cell tumor (GCT) cells as there is no consensus on which solution leads to the greatest amount of cell death. An in vitro evaluation was performed by exposing human GCT and human chondrosarcoma cell lines to 0.9% saline solution, sterile water, 70% ethanol, 3% hydrogen peroxide, 0.05% chlorhexidine gluconate (CHG), and 0.3% povidone iodine solutions independently for 2 and 5 minutes. A low-cytotoxicity control (LCC) and a high-cytotoxicity control (HCC) were established to determine the mean cytotoxicity of each solution and each solution's superiority to LCC and non-inferiority to HCC. The present study demonstrated that 0.05% CHG was non-inferior to the HCC when chondrosarcoma was exposed for 5 minutes and when GCT was exposed for 2 and 5 minutes (mean cytotoxicity, 99% to 102%) (p < 0.003 for all). Sterile water was superior to the LCC when chondrosarcoma was exposed for 5 minutes and when GCT was exposed for 2 minutes (mean, 28% to 37%) (p < 0.05). Sterile water (mean, 18% to 38%) (p < 0.012) and 3% hydrogen peroxide (mean, 7% to 16%) (p < 0.001) were both inferior to the HCC. The 3 other solutions were non-superior to the LCC (mean, -24% to -5%) (p < 0.023). In vitro irrigation in 0.05% CHG provided high cytotoxicity, comparable with the HCC. Therefore, the use of a 0.05% CHG solution clinically could serve as a potential chemical adjuvant during intralesional curettage of chondrosarcoma and GCT. In an effort to reduce the burden of residual tumor cells, irrigation solutions are often utilized as adjuvant local therapy. Use of a 0.05% CHG solution clinically could serve as a potential chemical adjuvant to intralesional curettage of chondrosarcoma and GCT. Further in vivo studies may be indicated to assess clinical outcomes and safety associated with the use of 0.05% CHG in the treatment of chondrosarcoma and GCT.

Cellular and Molecular Biological Alterations after Photon, Proton, and Carbon Ions Irradiation in Human Chondrosarcoma Cells Linked with High-Quality Physics Data.

Chondrosarcomas are particularly difficult to treat due to their resistance to chemotherapy and radiotherapy. However, particle therapy can enhance local control and patient survival rates. To improve our understanding of the basic cellular radiation response, as a function of dose and linear energy transfer (LET), we developed a novel water phantom-based setup for cell culture experiments and characterized it dosimetrically. In a direct comparison, human chondrosarcoma cell lines were analyzed with regard to their viability, cell proliferation, cell cycle, and DNA repair behavior after irradiation with X-ray, proton, and carbon ions. Our results clearly showed that cell viability and proliferation were inhibited according to the increasing ionization density, i.e., LET, of the irradiation modes. Furthermore, a prominent G2/M arrest was shown. Gene expression profiling proved the upregulation of the senescence genes CDKN1A (p21), CDKN2A (p16NK4a), BMI1, and FOXO4 after particle irradiation. Both proton or C-ion irradiation caused a positive regulation of the repair genes ATM, NBN, ATXR, and XPC, and a highly significant increase in XRCC1/2/3, ERCC1, XPC, and PCNA expression, with C-ions appearing to activate DNA repair mechanisms more effectively. The link between the physical data and the cellular responses is an important contribution to the improvement of the treatment system.

High-Resolution Secretome Analysis of Chemical Hypoxia Treated Cells Identifies Putative Biomarkers of Chondrosarcoma.

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma.

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

BackgroundAlthough 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.MethodsThe 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.ResultsChondrosarcoma 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.ConclusionsThese data demonstrated the significant anti-tumorigenic effect of shikonin derivatives in chondrosarcoma and encourage further research.

Abstract 1812: Therapeutic targeting and development of IDH inhibitors for the treatment of chondrosarcoma

Abstract Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Recurrent somatic mutations in the isocitrate dehydrogenase I (IDH1) genes have been identified in a spectrum of human malignancies, including chondrosarcoma. Mutations in these genes lead to impaired ability of IDH1 to catalyze the conversion of isocitrate to alpha ketoglutarate and to gain of a neomorphic enzymatic activity which results in production of oncometabolite 2-hydroxyglutarate (2HG). Our preliminary data indicated that compared to samples that were wild type, IDH1 mutant samples showed a significant increase in intra-tumoral 2HG levels. In the present study, we hypothesized that 2-HG accumulation induces DNA and histone hypermethylation and altered gene expression, ultimately resulting in a block in cellular differentiation by competitively inhibiting α-ketoglutarate-dependent dioxygenases involved in histone and DNA demethylation. Therefore, we proposed that inhibition of IDH should mediate its antitumor effects through the induction of differentiation. To test this hypothesis, we utilized human chondrosarcoma cell lines including IDH wild type (CH2879) and IDH1 mutant (JJ012) and the IDH1 inhibitor FT6535. Results from our in vitro cell viability assay and histone methylation showed that selective inhibition of IDH1 resulted in modest inhibition of cell proliferation and suppression of histone methylation in IDH1 mutant chondrosarcoma cells. Next we elected to test the IDH inhibitor in combination with differentiating agents including retinoic acid. Cell viability assays showed that when compared to either agent alone, the combination of retinoic acid and FT6535 resulted in a significant decrease in cellular proliferation. Western blot analysis showed induction of COL1A2 and SOX9 (differentiation markers) with suppression of histone methylation H3K27me3 or H3K9me3, Taken together, our data strongly suggests that combination treatment with the IDH1 inhibitor FT6535 and retinoic acid represents a novel approach to suppress cell growth by epigenetic modulation to induce differentiation and merits further evaluation as a potential treatment modality in chondrosarcoma. Citation Format: Tahir N. Sheikh, Chao Lu, Gary K. Schwartz. Therapeutic targeting and development of IDH inhibitors for the treatment of chondrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1812.

RNA binding protein GNL3 up-regulates IL24 and PTN to promote the development of osteoarthritis

Osteoarthritis (OA) is a degenerative disease, which has a high incidence in middle-aged and elderly people and tends to occur in weight-bearing or active joints. Current treatment can only relieve symptoms and delay the progression of OA in result of its indistinct pathogenesis. In recent years, more and more studies have focused on the pathogenesis of OA. Nucleolar GTP binding protein 3 (GNL3) is associated with chondrogenic differentiation and can participate in genomic regulation as RNA binding protein (RBP). We used RNA sequencing (RNA-seq) to analyze the overall transcription level of the human cervical cancer cell line HeLa after GNL3 deletion. The results showed that downstream genes IL24 and PTN were down-regulated. IL24 takes part in the progression of OA by inducing articular osteocyte apoptosis, while PTN conducts to the progression of OA by promoting angiogenesis. We validated the results in the human chondrosarcoma cell line SW1353 and OA patients. Compared with the control group, GNL3, IL24 and PTN genes were elevated in OA specimens. This study explored the relationship between GNL3 and these two downstream genes, hoping to find biomarkers in the pathogenesis of osteoarthritis that can be used as therapeutic targets in the future.

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.

Effects of miR-146a-5p on chondrocyte interleukin-1β-induced inflammation and apoptosis involving thioredoxin interacting protein regulation.

ObjectiveOsteoarthritis (OA) is a chronic degenerative arthropathy characterized by articular cartilage degeneration, subchondral osteosclerosis, and hyperosteogeny. MicroRNAs (miRNAs) play an important regulatory role in its pathological development, so this study explored the effect and potential mechanism of miR-146a-5p in interleukin (IL)-1β-induced OA cartilage injury.MethodsThe human chondrosarcoma cell line SW1353 and normal human chondrocytes C28/I2 were stimulated by IL-1β to construct the OA chondrocyte model. miR-146a-5p and thioredoxin interacting protein (TXNIP) expression levels were detected by quantitative real-time (qRT)-PCR and western blot. Their expression was modified by transfecting an miR-146a-5p inhibitor, mimic, and pcDNA-TXNIP. The relationship between miR-146a-5p and TXNIP was analyzed by the dual-luciferase assay, while cell viability, apoptosis, and inflammatory expression were determined by cell counting, TUNEL staining, and ELISA, respectively.ResultsmiR-146a-5p expression was upregulated in SW1353 and C28/I2 cells stimulated by IL-1β. miR-146a-5p knockdown significantly enhanced cell activity, inhibited inflammatory factor expression, and reduced cell apoptosis. The dual-luciferase assay revealed TXNIP as a target gene of miR-146a-5p and suggested that miR-146a-5p promotion of OA damage could be reversed by upregulating TXNIP.ConclusionThese results suggest that miR-146a-5p inhibits cell proliferation and promotes apoptosis and the inflammatory response in OA cartilage injury by modulating TXNIP.

Chondrosarcoma-derived Mutation in IDH1 Promotes Tumorigenesis by Inducing HIF-1α

IDH mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into an oncometabolite, D-2-hydroxyglutarate (D-2HG). IDH mutations occur in more than 50% of patients with conventional or dedifferentiated chondrosarcoma. However, the function of these mutations in the pathogenesis of chondrosarcoma is still unclear. In this study, we aimed to establish a mutant IDH1 knockout model in a chondrosarcoma cell line and determine the role of mutant IDH1 in this malignancy. In our study, we employed a human chondrosarcoma cell line, JJ012 which harbors an endogenous IDH1R132G mutation. As a control, we also used an immortalized chondrocyte cell line, C28 which expresses wildtype IDH1 only. IDH1 was knocked out via CRISPR/Cas9 technology. Quantitative analyses of D-2HG were conducted by tandem mass spectrometry. RNA-Seq analysis was performed to identify differentially expressed genes between the mutant IDH1 knockout (KO) cells and the parental cells. RNA-Seq data were validated by real time PCR. Colony-forming ability of KO cells under hypoxia and normoxia conditions was evaluated using soft agar assay. The effect of mutant IDH1 on chondrosarcoma growth was analyzed in murine xenograft models. The expression of HIF1-1α and its targeted genes were examined in cells and tumor tissues by western blot and Immunohistochemistry (IHC). Mutant IDH1 was successfully knocked out in JJ012 cells. Single cell-derived KO clones were then identified and developed. As expected, D-2HG production is almost completely depleted in these KO clones. Using these genetically modified cells, we found that knockout of mutant IDH1 remarkably inhibited colony formation by 40% under normoxia condition compared to the parental control (p<0.05). Intriguingly, we observed that the HIF1-α levels were significantly reduced in the JJ012 KO cells compared with the parental cells. Moreover, RNA-seq data showed a widespread downregulation of HIF1-1α targeted genes such as VEGFA, VEGFC, SLC2A3, EDN1 in response to the knockout (FDR-adjusted p value < 0.01). These data were confirmed by qRT-PCR. The soft-agar colony formation assay further showed a significantly increased colony number and diameter for JJ012 KO cells under hypoxia condition compared with those under normoxia condition (P<0.05). Furthermore, we observed that loss of mutant IDH1 led to a marked attenuation of chondrosarcoma formation in the murine xenograft model. Immunoblotting and IHC revealed that HIF-1α and its targeted genes were dramatically downregulated in the KO cell-derived tumors. We found that mutant IDH1 contributes to tumorigenicity of chondrosarcoma cells, at least partially through the upregulation of HIF1-α. This suggests that targeting HIF1-α signaling pathway is promising for combinatorial regimens with mutant IDH inhibitors for patients with IDH1-mutant chondrosarcoma.

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.

CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage.

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.

Lumican mediates HTB94 chondrosarcoma cell growth via an IGF‑IR/Erk1/2 axis.

Chondrosarcoma is a malignant bone tumor characterized by the production of a modified cartilage-type extracellular matrix (ECM). In the present study, the expression levels of the small leucine-rich proteoglycans (SLRPs), decorin, biglycan and lumican, were examined in the HTB94 human chondrosarcoma cell line. HTB94 cells were found to express and secrete the 3 SLRP members. RT-qPCR and western blot analysis demonstrated that lumican was the most abundantly secreted SLRP, whereas decorin and biglycan expression levels were low. The utilization of short interfering RNA specific for the decorin, biglycan, and lumican genes resulted in the efficient downregulation of the respective mRNA levels (P≤0.001). The growth of the HTB94 cells was stimulated by lumican (P≤0.001), whereas their migration and adhesion were not affected (P=NS). By contrast, these cellular functions were not sensitive to a decrease in low endogenous levels of decorin and biglycan. Lumicandeficiency significantly inhibited both basal and insulin-like growth factor I (IGF-I)-induced HTB94 cell growth (P≤0.001 andP≤0.01, respectively). These effects were executed through the insulin-like growth factor I receptor (IGF-IR), whose activation was markedly attenuated (P≤0.01) in lumican-deficient HTB94 cells. The downregulation of lumican induced the substantial inhibition of extracellular regulated kinase (ERK1/2) activation (P≤ 0.01), indicating that ERK1/2 is a necessary component of lumican/IGF-IR-mediated HTB94 cell proliferation. Moreover, the lumican-deficient cells exhibit increased mRNA levels of p53 (P≤0.05), suggesting that lumican facilitates HTB94 cell growth through an IGF-IR/ERK1/2/p53 signaling cascade. On the whole, the findings of the present study demonstrate that endogenous lumican is a novel regulator of HTB94 cell growth.

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.