Human Chondrosarcoma Cell Line Research Articles

Regulation of onco and tumor suppressor MiRNAs by mTORC1 inhibitor PRP-1 in human chondrosarcoma

Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent. This study aimed to reveal the comparative analysis of miRNAs and their targets in human JJ012 chondrosarcoma cell line between control and experimental samples, treated with mTORC1 inhibitor, cytostatic antiproliferative proline-rich polypeptide (PRP-1). Examination of tumor-specific microRNA expression profiles has revealed widespread deregulation of these molecules in diverse cancers. It was reported that microRNAs can function as novel biomarkers for disease diagnostics and therapy, as well as a novel class of oncogenes and tumor suppressor genes. mTORC 1 inhibitor PRP-1 caused significant upregulation of tumor suppressors, such as miR20a, miR125b, and miR192; and downregulation of onco miRNAs, miR509-3p, miR589, miR490-3p, miR 550 in human chondrosarcoma JJ012 cell line.

Gallic acid induces apoptosis and inhibits cell migration by upregulating miR-518b in SW1353 human chondrosarcoma cells

Gallic acid (GA), a natural agent, is widely distri-buted in plants with a range of biological effects and has been of potential interest as anticancer agent. However, its effects on chondrosarcoma cell apoptosis are still undefined. In the present study, the possible mechanisms of GA-induced apoptosis were explored in SW1353 cells, a human chondrosarcoma cell line. Our results showed that GA inhibited cell viability dose- and time-dependently. Morphological examination of GA-treated cells exhibited the typical features of cell death, such as rounding up of the cells and cell shrinkage. Wound-healing assay indicated that GA inhibited the migratory abilities of SW1353 cells. Hoechst 33258 staining assay and Annexin V/PI staining assay exhibited apoptosis induction by GA. To determine the molecular mechanism of GA-induced apoptosis, the expression levels Bcl-2, Bax, caspase-3 and caspase-9 were determined in SW1353 cells treated with GA. We found that GA downregulated the expression of the anti-apoptotic protein Bcl-2, and upregulated the expression of the pro-apoptotic protein Bax, and the activation of caspase-3 and caspase-9. To identify the possible mechanisms, the changes of microRNA expression were tested using the miRCURY™ LNA expression array. It was observed that the miR-518b gene was upregulated in treated cells. Taken together, these data show that GA induces apoptosis and inhibits cell migration by upregulating miR-518b in SW1353 cells.

Apoptosis Signal-Regulating Kinase 1 Is Involved in Brain-Derived Neurotrophic Factor (BDNF)-Enhanced Cell Motility and Matrix Metalloproteinase 1 Expression in Human Chondrosarcoma Cells

Chondrosarcoma is the primary malignancy of bone that is characterized by a potent capacity to invade locally and cause distant metastasis, and is therefore associated with poor prognoses. Chondrosarcoma further shows a predilection for metastasis to the lungs. The brain-derived neurotrophic factor (BDNF) is a small molecule in the neurotrophin family of growth factors that is associated with the disease status and outcome of cancers. However, the effect of BDNF on cell motility in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma cell lines had significantly higher cell motility and BDNF expression compared to normal chondrocytes. We also found that BDNF increased cell motility and expression of matrix metalloproteinase-1 (MMP-1) in human chondrosarcoma cells. BDNF-mediated cell motility and MMP-1 up-regulation were attenuated by Trk inhibitor (K252a), ASK1 inhibitor (thioredoxin), JNK inhibitor (SP600125), and p38 inhibitor (SB203580). Furthermore, BDNF also promoted Sp1 activation. Our results indicate that BDNF enhances the migration and invasion activity of chondrosarcoma cells by increasing MMP-1 expression through a signal transduction pathway that involves the TrkB receptor, ASK1, JNK/p38, and Sp1. BDNF thus represents a promising new target for treating chondrosarcoma metastasis.

CXCR4-Targeted Therapy Inhibits VEGF Expression and Chondrosarcoma Angiogenesis and Metastasis

Chondrosarcoma is notable for its lack of response to conventional cytotoxic chemotherapy, propensity for developing lung metastases, and poor survival. Therefore, a better understanding of angiogenic and metastatic pathways is needed. Multiple pathways regulate angiogenesis and metastasis, including chemokines and their receptors. In this study, we investigated chemokine (C-X-C motif) receptor 4 (CXCR4) signaling in chondrosarcoma and tested the hypotheses that CXCR4 inhibition suppresses tumor angiogenesis and metastasis. CXCR4 expression, analyzed by real-time PCR and Western blot, was increased in human chondrosarcoma cell line JJ compared with normal chondrocytes and was further increased in JJ by hypoxia (2% O2), vascular endothelial growth factor A (VEGFA; 10 ng/mL), and in xenograft tumors in nude mice. The CXCR4 ligand CXCL12 (10 ng/mL) doubled secreted VEGFA, measured with ELISA, under hypoxic conditions and this conditioned media increased human umbilical vein endothelial cell tube formation. These effects were inhibited by CXCR4 siRNA or AMD3100 (5 μg/mL). In a xenograft mouse model, four weeks of AMD3100 treatment (1.25 mg/kg, intraperitoneally twice daily) inhibited tumor angiogenesis, tumor growth, and metastasis. VEGFA content in tumor extracts was decreased (7.19 ± 0.52 ng/mL control vs. 3.96 ± 0.66 treatment) and bioimaging of angiogenesis was decreased by 56%. Tumor volumes averaged 4.44 ± 0.68 cm(3) in control compared with 2.48 ± 0.61 cm(3) in the treatment group. The number of lung metastatic nodules was 23 ± 9 in control compared with 10 ± 6 in the treatment group (N = 8/group). Therefore, CXCR4-targeted therapy may be a treatment strategy for chondrosarcoma.

Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress.

Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in adult human sarcoma cell lines

Adult sarcomas are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the ECM and basement membrane, allowing cancer cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on the activity of u-PA, MMPs and TIMPs in various human adult sarcomas. Human fibrosarcoma (HT-1080), chondrosarcoma (SW-1353), liposarcoma (SW-872), synovial sarcoma (SW-982) and uterine leimyosarcoma (SK-UT-1) cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1,000 μg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Fibrosarcoma, chondrosarcoma, liposarcoma and leiomyosarcoma cancer cell lines expressed u-PA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to u-PA were detected for synovial sarcoma cells. On gelatinase zymography, fibrosarcoma, chondrosarcoma, liposarcoma and synovial sarcoma showed bands corresponding to MMP-2 and MMP-9 with enhancement of MMP-9 with PMA (100 ng/ml) treatment. Uterine leiomyosarcoma showed strong bands corresponding to inactive and active MMP-9 and a faint band corresponding to MMP-9 dimer induced with PMA treatment, but no MMP-2 band. NM inhibited their expression in a dose-dependent manner. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in treatment of adult sarcomas.

Abstract 3777: Low physiological fluid shear exposure induces metastatic potential of chondrosarcoma cells via MMP-12 activation.

Abstract Interstitial flow in and around tumor tissue affects the mechanical microenvironment to modulate tumor cell metastasis. We investigated the roles of flow-induced shear stress in modulating chondrosarcoma cell metastatic potential such as cell invasiveness and motility, and the underlying mechanisms. Using human chondrosarcoma cell lines as a model system, we demonstrated that low fluid shear (2 dyn/cm2), but not high fluid shear (20 dyn/cm2), exposure for 48 h remarkably induced MMP-12 mRNA by ∼35-fold and significantly triggered it proteolytic activity among all MMP members. The molecular basis of the shear effect was further analyzed, and our data revealed that low fluid shear stress induces the rapid synthesis of VEGF-B, VEGF-D and IGF-2, which in turn transactivates MMP-12 expression via PI3K and p38 MAPK-dependent pathways. Furthermore, highly induced MMP-12 markedly enhanced chondrosarcoma cell invasion and motility by using chemtaxis transwell chambers in conjunction with time-lapse micro-channel assays. Collectively, our data suggest that the induction of IGF-2, VEGF-B, and VEGF-D under low fluid shear, which in turn upregulates MMP-12 via PI3K and p38 MAPK signaling pathways, leading to the enhanced invasive and migratory capabilities of chondrosarcoma cells. Our findings provide insights into the mechanism by which shear stress induces chondrosarcoma metastatic potential and demonstrate the importance of mechanical environments in modulating molecular signaling, gene expression and functions in tumor cells. Citation Format: Shih-Hsun Chen, Pu Wang, Wei-Chien Hung, Konstantinos Konstantopoulos. Low physiological fluid shear exposure induces metastatic potential of chondrosarcoma cells via MMP-12 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3777. doi:10.1158/1538-7445.AM2013-3777

1-Benzyl-2-Phenylbenzimidazole (BPB), a Benzimidazole Derivative, Induces Cell Apoptosis in Human Chondrosarcoma through Intrinsic and Extrinsic Pathways

In this study, we investigated the anticancer effects of a new benzimidazole derivative, 1-benzyl-2-phenyl -benzimidazole (BPB), in human chondrosarcoma cells. BPB-mediated apoptosis was assessed by the MTT assay and flow cytometry analysis. The in vivo efficacy was examined in a JJ012 xenograft model. Here we found that BPB induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353) but not in primary chondrocytes. BPB induced upregulation of Bax, Bad and Bak, downregulation of Bcl-2, Bid and Bcl-XL and dysfunction of mitochondria in chondrosarcoma. In addition, BPB also promoted cytosolic releases AIF and Endo G. Furthermore, it triggered extrinsic death receptor-dependent pathway, which was characterized by activating Fas, FADD and caspase-8. Most importantly, animal studies revealed a dramatic 40% reduction in tumor volume after 21 days of treatment. Thus, BPB may be a novel anticancer agent for the treatment of chondrosarcoma.

Curcumin induces cell apoptosis in human chondrosarcoma through extrinsic death receptor pathway

Chondrosarcoma is a soft tissue sarcoma with a poor prognosis that is unresponsive to conventional chemotherapy. Surgical treatment leads to severe disability with high rates of local recurrence and life threat. Curcumin, an active compound in turmeric and curry, has been proven to induce tumor apoptosis and inhibit tumor proliferation, invasion, angiogenesis, and metastasis of cancer cells. In this study, we investigated the anticancer effects of curcumin in human chondrosarcoma cells. Curcumin induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353) but not in primary chondrocytes. Curcumin induced upregulation of Fas, FasL, and DR5 expression in chondrosarcoma cells. Transfection of cells with Fas, FasL, or DR5 siRNA reduced curcumin-induced cell death. In addition, p53 involved in curcumin-mediated Fas, FasL, and DR5 expression and cell apoptosis in chondrosarcoma cells. Most importantly, animal studies revealed a dramatic 60% reduction in tumor volume after 21days of treatment. Thus, curcumin may be a novel anticancer agent for the treatment of chondrosarcoma.

Lutein and β-Cryptoxanthin Inhibit Inflammatory Mediators in Human Chondrosarcoma Cells Induced with IL-1β

Objective: Studies have shown that lutein (Lu) and β-cryptoxanthin (βCr) may down-regulate factors involved in inflammation associated with osteoarthritis and rheumatoid arthritis. We studied the possible protective effects of Lu and βCr in vitro against human chondrocyte dysfunction using a human chondrosarcoma cell line. Methods: SW-1353 human chondrosarcoma cells were cultured for 24 hr in supplemented medium containing 0, 0.01, 0.1 or 1.0 µmol/L of Lu or βCr and subsequently stressed for 24 hr in the presence of 10 µg/L IL-1β. The resulting condi- tioned medium was analyzed for matrix-metalloproteinase-13 (MMP-13), cytokines (IL-1α, IL-2, IL-4, IL-10, IFN-γ , IL- 6, IL-8, and TNF-α), and PGE2. Nuclear extract from the harvested cells was analyzed for NFκB. Results: Lu (1.0 µmol/L; P<0.05) but not βCr decreased MMP-13. Both Lu (1.0 µmol/L; P<0.05) and βCr (0.1 and 0.01 µmol/L; P<0.01) inhibited PGE 2 production. All concentrations of βCr suppressed (P<0.05) IL-1α, IL-2 and IFN-γ pro- duction while Lu increased concentrations of these cytokines. Lu increased (P<0.05) while βCr decreased IL-4 and IL-10 concentrations. NFκB p50 production was suppressed (P<0.01) by both Lu and βCr, with Lu being more inhibitory. Conclusion: Therefore, Lu and βCr protected against IL-1β-induced chondrocyte dysfunction by down-regulating NFκB activation and inhibiting inflammatory response, albeit through somewhat different pathways.

Interleukin-1β induced nuclear factor-κB binds to a disintegrin-like and metalloproteinase with thrombospondin type 1 motif 9 promoter in human chondrosarcoma cells.

Nuclear factor-κB (NF-κB) is involved in the regulation of inflammation‑associated genes. NF-κB forms dimers which bind with sequences referred to as NF-κB sites (9-11 bp). A disintegrin-like and metalloproteinase with thrombospondin type 1 motif 9 (ADAMTS9) is a type of proteoglycanase, which proteolytically cleaves versican and aggrecan. ADAMTS9 is a cytokine-inducible gene that contains binding sites for NF-κB within its promoter region. Interleukin-1β (IL-1β) affects cartilage metabolism and is involved in the NF-κB pathway. It is therefore hypothesized that NF-κB binding with ADAMTS9 promoters may activate IL-1β, thereby promoting chondrocytic cell growth. In the present study, the OUMS-27 chondrocytic human chondrosarcoma cell line was treated with IL-1β with or without inhibitors of NF-κB signaling pathways. Chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA) were conducted order to analyze the binding of NF-κB with the ADAMTS9 promoter region. NF-κB-p65 subunit phosphorylation was promoted in IL-1β-treated cells, which were not treated with inhibitors of NF-κB signaling pathways. By contrast, NF-κB-p65 subunit phosphorylation was inhibited in cells that had been treated with BAY-117085, an NF-κB pathway inhibitor. ChIP and EMSA assays demonstrated that, following treatment with IL-1β, NF-κB‑p65 bound to elements located at -1177 and -1335 in the ADAMTS9 promoter region, in contrast to the untreated samples. The results of the present study suggested that NF-κB may be involved in IL-1β-induced activation of ADAMTS9 in human chondrocytes.

Bone and Joint Protection Ability of Ceramic Material with Biological Effects

Ceramic materials with biological effects (bioceramic) have been found to modulate various biological effects, especially those effects involved in antioxidant activity and hydrogen peroxide scavenging. As arthropathy and osteopathy are the major chronic diseases of geriatric medicine, we explored the possible activity of bioceramic on these conditions using animal and cell models. Rabbits received intra-articular injections of lipopolysaccharides (LPS) to induce inflammation that mimic rheumatic arthritis. FDG isotopes were then IV injected for PET scan examinations at 16 hours and 7 days after the LPS injection. We examined and compared the bioceramic and control groups to see if bioceramic was capable of relieving inflammation in the joints by subtracting the final and initial uptake amount of FDG (max SUV). We studied the effects in prostaglandin E2 (PGE2) inhibition on the human chondrosarcoma (SW1353) cell line, and the effects on the murine osteoblast (MC3T3-E1) cell line under oxidative stress. All the subtractions between final and initial uptakes of FDG in the left knee joints of the rabbits after LPS injection indicated larger decreases in the bioceramic group than in the control group. This anti-arthritic or inflammatory effect was also demonstrated by the PGE2 inhibition of the SW1353 cells. We further proved that bioceramic treatment of the MC3T3-E1 cells resulted in increased viability of osteoblast cells challenged with hydrogen peroxide toxicity, and increased alkaline phosphatase activity and the total protein production of MC3T3-E1 cells under oxidative stress. Since LPS-induced arthritis is an experimental model that mimics RA, the potential therapeutic effects of bioceramic on arthropathy merit discussion. Bioceramic may contribute to relieving inflammatory arthritis and maintaining bone health.

Incorporation of exudates of human platelet‐rich fibrin gel in biodegradable fibrin scaffolds for tissue engineering of cartilage

The goal of this study was to assess the incorporation of exudates of human platelet-rich fibrin (hPRF) that is abundant in platelet cytokines and growth factors into biodegradable fibrin (FB) scaffolds as a regeneration matrix for promoting chondrocyte proliferation and re-differentiation. hPRF was obtained from human blood by centrifugation without an anticoagulant, and the exudate of hPRF was collected and mixed with bovine fibrinogen, and then thrombin was added to form the FB scaffold. Proliferation and differentiation of human primary chondrocytes and a human chondrosarcoma cell line, the SW-1353, embedded in the three-dimensional (3D) scaffolds and on the two-dimensional (2D) surface of the FB scaffolds so produced were evaluated in comparison with an agarose (AG) scaffold serving as the control. Results demonstrated that the amounts of these cytokines and growth factors in hPRF exudates were higher than those in the blood-derived products except for TGF-β1. Chondrocytes and SW1353 cells on the 2D and 3D FB scaffolds with the addition of the exudates of PRF exhibited more-available proliferation and differentiation than cells on 2D and 3D FB and AG scaffolds. It was concluded that FB scaffolds can provide an appropriate environment for chondrocyte proliferation and re-differentiation, and it could be improved by adding exudates of hPRF. These 3D scaffolds have great promise for cartilage tissue engineering.

MTORC1 inhibition and ECM–cell adhesion-independent drug resistance via PI3K–AKT and PI3K–RAS–MAPK feedback loops

Mammalian target of rapamycin (mTOR) serine threonine kinase is the enzyme that regulates cancer cell growth by altering nutrient supplies to cancer cells. The neuropeptide (proline-rich peptide 1 (PRP-1)), galarmin, produced by the brain neurosecretory cells is a mTOR kinase inhibitor with powerful 80% antiproliferative cytostatic effect in a high-grade chondosarcoma and other mesenchymal tumors. However, the negative feedback loop of phosphatidylinositol 3 kinase-Protein kinase B (PKB), PI3K-AKT and PI3K-rat sarcoma (RAS)-mitogen-activated protein kinase (MAPK) activation is well documented for mTOR inhibitors. This study explored the involvement of those loops in drug resistance after the treatment with mTOR complex 1 (mTORC1) inhibitor, PRP-1. Multidrug resistance assay (MDR) demonstrated that this cytokine did not inhibit permeability glycoprotein-mediated MDR in chondrosarcoma. Phospho-MAPK array in human chondrosarcoma cell line treated with galarmin (10μg/ml,) showed a strong upregulation of phosphorylated glycogen synthase kinase 3β (GSK3β) via activation of PI3K-AKT and MAPK feedback loops. Such GSK3β inactivation leads to β-catenin accumulation that entails drug resistance. The ability of cells to metastasize is reflected in their capacity to adhere to extracellular matrix and endothelium. Laminin cell adhesion assay demonstrated that PRP-1 in the same concentrations that inhibit mTOR kinase inhibited JJ012 chondrosarcoma cell adhesion. The neuropeptide did not have any effect on the expression of total focal adhesion kinase and its phosphorylated form. Thus, it was not accompanied by total HAT downregulation and total HDAC upregulation. Combinatorial treatments of PRP-1 with MAPK and PI3K/AKT inhibitors most probably will lead to full cytotoxicity overcoming drug resistance.

A novel orthotopic animal model of human chondrosarcoma

SummaryChondrosarcoma is the second most common primary malignant bone tumour in humans. Currently, surgical resection is the only appropriate curative approach as it is relatively unresponsive to traditional chemoand radiotherapy. However, a complete resection is often hindered due to the proximity to organs resulting in a poor outcome of this challenging malignancy. Few novel antitumour agents have been tested on different chondrosarcoma cell lines in vitro so far. In order to qualify new agents in vivo, animal models are often used in which cell lines are subcutaneously injected prior to chemotherapeutical treatment. These types of models often lack relevance to the human chondrosarcoma as the number of agents that fail in the clinic far outweighs those considered effective on in vivo studies. Orthotopic xenograft models however are of much more predictive value. Thus, the development of a novel orthotopic animal model for human chondrosarcoma using a three-dimensional matrix carrying tumour cells, was the aim of this study. For that purpose, SW-1353, a human bone chondrosarcoma cell line, was first cultured in MatrigelTM, followed by orthotopic implantation into10 SCID mice by intra-tibial injection. After 40 days, the animals developed localized bone tumours verified by radiographic and histological examinations. Radiologic and histological sections showed osteolysis and invasive tumour growth. This study demonstrates a promising new method for effective and reproducible orthotopic implantation of human chondrosarcoma. The presented animal model allows further examination and can be used as a predictive preclinical model for anticancer drug activity in humans.

A Role for Prolyl 3-Hydroxylase 2 in Post-translational Modification of Fibril-forming Collagens

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.

FPipTB, a benzimidazole derivative, induces chondrosarcoma cell apoptosis via endoplasmic reticulum stress and apoptosis signal‐regulating kinase 1

Chondrosarcoma is the second most common primary bone tumor and it responds poorly to both chemotherapy and radiation treatment. In this study, we investigated the anticancer effects of a new benzimidazole derivative, 2-(furanyl)-5-(piperidinyl)- (3,4,5-trimethoxybenzyl) benzimidazole (FPipTB) in human chondrosarcoma cells. FPipTB-induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353) but not in primary chondrocytes. Furthermore, it triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels. Treatment of chondrosarcoma cells with FPipTB was associated with increased intracellular levels of ASK1, p38, p53, and Bax, followed by release of cytochrome c from mitochondria and activation of caspases. It is also known that ER stress activates apoptosis signal-regulating kinase 1 (ASK1), which mediates activation of JNK and p38 pathways. We also found that FPipTB-induced p38 and p53 phosphorylation and upregulated Bax expression. To study the mechanism of Bax upregulation, we determined that Bax promoter activity was increased in FPipTB-treated cells, leading to an increase in intracellular levels of Bax. In addition, cell treated with Ca(2+) chelator or p38 inhibitor showed reduced transcriptional activity. The results further suggest that FPipTB triggered ER stress, as indicated by changes in cytosolic calcium levels and activated the ASK1-MKK3/6-p38-p53-Bax pathway, causing chondrosarcoma cell death. Importantly, animal studies revealed a dramatic 40% reduction in tumor volume after 21 d of treatment. Thus, FPipTB may be a novel anticancer agent for the treatment of chondrosarcoma.

Epigallocatechin-3-gallate induces cell apoptosis of human chondrosarcoma cells through apoptosis signal-regulating kinase 1 pathway

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. The aim of this study was to elucidate the mechanism of EGCG-induced apoptosis of human chondrosarcoma cells. EGCG induced cell apoptosis in human chondrosarcoma cell lines but not primary chondrocytes. EGCG induced upregulation of Bax and Bak, downregulation of Bcl-2 and Bcl-XL, and dysfunction of mitochondria in chondrosarcoma. We also found that the accumulation of reactive oxygen species (ROS) is a critical mediator in EGCG-induced cell death. EGCG induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of chondrosarcoma cells with EGCG induced p38 and c-jun-NH2-kinase (JNK) phosphorylation. Transfection with ASK1 siRNA or p38 and JNK mutant antagonized the EGCG-induced cell apoptosis. Therefore, EGCG triggered ROS and activated the ASK1-p38/JNK pathway, resulting chondrosarcoma cell death. Importantly, animal studies revealed a dramatic reduction in tumor volume after 24 days of treatment. Thus, EGCG may be a novel anti-cancer agent for the treatment of chondrosarcoma.

Quaternary ammonium-melphalan conjugate for anticancer therapy of chondrosarcoma: in vitro and in vivo preclinical studies

Cartilage tumours present ongoing therapeutic challenges due to their chondrogenic extracellular matrix that potentially hampers drug delivery, their low percentage of dividing cells, and their poor vascularity. In this context, and based on the affinity of the quaternary ammonium moiety for proteoglycans (PG), we developed a strategy that uses the quaternary ammonium function to selectively deliver DNA alkylating agents to the cartilage tumour tissue. We engineered the quaternary ammonium derivative of melphalan (Mel-AQ) and assessed its antitumoural activity in vitro and in vivo. In vitro, micromolar concentrations of Mel-AQ inhibited the proliferation of human HEMC-SS chondrosarcoma and Saos-2 osteosarcoma cell lines. Moreover, 24-h incubation with 20μM Mel-AQ induced a 2.5-fold increase in S population and a 1.5-fold increase in subG0G1 population compared to controls. In vivo, Mel-AQ demonstrated antitumour activity in the orthotopic model of primary Swarm rat chondrosarcoma. When given to chondrosarcoma-bearing rats (three doses of 16 μmol/kg at days 8, 12 and 16 post-implant), Mel-AQ demonstrated an optimal antitumour effect at day 43, when tumour cell growth inhibition peaked at 69%. Interestingly, the treatment protocol was proved well tolerated, since the animals showed no weight loss over the course of the study. This antitumoural effect was assessed in vivo by scintigraphic imaging using (99m)Tc-NTP 15-5 developed in our lab as a PG-targeting radiotracer, and tumour tissue was analyzed at study-end by biochemical PG assay with Alcian blue staining. Mel-AQ treatment led to a significant decrease in the PG content of tumoural tissue. These experimental results highlighted the promising antitumour potential of Mel-AQ as a PG-targeting strategy for therapeutic management of chondrosarcoma.

α-Mangostin Induces Apoptosis in Human Chondrosarcoma Cells through Downregulation of ERK/JNK and Akt Signaling Pathway

Chondrosarcoma is a malignant primary bone tumor that is resistant to chemotherapy and radiation therapy. α-Mangostin, a component of Garcinia mangostana Linn, is a xanthone derivative shown to have antioxidant and antitumor properties. This study is the first to investigate anticancer effects of α-mangostin in the human chondrosarcoma cell line SW1353. We showed that α-mangostin inhibited cell proliferation of SW1353 cells in a time- and dose-dependent manner by using the trypan blue exclusion method. Hoechst 33342 nuclear staining and nucleosomal DNA-gel electrophoresis revealed that α-mangostin could induce nuclear condensation and fragmentation, typically seen in apoptosis. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. α-Mangostin activated caspase-3, -8, -9 expression, decreased Bcl-2 and increased Bax. This promotes mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytoplasm. In addition, total and phosphorylated ERK and JNK were downregulated in α-mangostin-treated SW1353 cells but no changes in p38. α-Mangostin also decreased phosphorylated Akt without altering total Akt. These results suggest that α-mangostin inhinbited cell proliferation and induced apoptosis through downregulation of ERK, JNK and Akt signaling pathway in human chondrosarcoma SW1353 cells.