Analysis of IL6‑protein complexes in chondrosarcoma
- Karina Galoian
- Shihua Luo
- Parthik Patel
- Published online on: November 10, 2017 https://doi.org/10.3892/br.2017.1016
Chronic inflammation and cytokines serve important roles in cancer. Cytokines are secreted proteins that are involved in immune response, cell communications and, depending on cellular context, may be anti or pro-tumorigenic (1). Chondrosarcoma is a cancer of the cartilage with systemic involvement. It is a common primary bone malignancy, accounting for 25% of bone sarcomas (2). Tumors typically develop in the pelvis, long bones and spine, as well as the larynx, head and neck (3). More aggressive forms of chondrosarcoma are characterized by early metastasis, and the metastasis rate of primary chondrosarcoma may reach 42%, and up to 86% in patients with local recurrence (4).
Chondrosarcoma does not typically respond to conventional therapies such as chemotherapy and radiation, which necessitates the identification of novel alternative therapies (2–7). The etiological factors and molecular pathways leading to the transformation of mesenchymal cells into sarcoma cells are unknown; therefore, understanding of the involvement of certain cytokines in failed differentiation programs leading to cancer or lost tumor suppressive functions is critical and of current interest (1). A previous study by our group reported from a human ELISA assay that the expression of interleukin 6 (IL6) in JJ012 chondrosarcoma cells was 86-fold lower than that in C28 chondrocytes, indicating it to be an anti-inflammatory and antitumorigenic factor (7). Furthermore, downregulation of IL6 has been reported for a number of tumor types, including undifferentiated thyroid carcinoma and thyroid cancer (8–10). To investigate IL6-protein interactions leading to these differences in IL6 expression, the present study assessed IL6 complexes in JJ012 and C28 cells through nuclear extraction and an electrophoretic mobility shift assay (EMSA), followed by 2D gel phoresis, in-gel trypsin digestion (11) and proteomic mass spectrometry (MS) analysis.
Materials and methods
Complete growth medium for human JJ012 chondrosarcoma cells were obtained from the laboratory of Dr Joel Block (Rush University Medical Centre, Chicago, IL, USA) and for C28 chondrocytes from the laboratory of Dr Sean Scully (University of Miami, Miami, FL, USA) comprised of the following: Dulbecco's modified Eagle's medium supplemented with F12, 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 25 µg/ml ascorbic acid, 100 ng/ml insulin, 100 nM hydrocortisone and 1% penicillin/streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cultures were incubated for 24 h in a humidified 5% CO2 incubator at 37°C.
Nuclear extraction and EMSA, and SDS-PAGE and IL6 oligonucleotide forward and reverse sequence synthesis
These procedures were performed as described in our recent study (7).
2D gel electrophoresis and in-gel trypsin digestion
The procedures were performed according to previously described methods (11) with slight modifications.
For the 2D gel electrophoresis (modified 2D EMSA), a gel shift EMSA kit (cat. no. 37341) from Active Motif, Inc. (Carlsbad, CA, USA) was used. The procedures from steps 1 to 5 as detailed in the EMSA kit manual were performed according to manufacturer's instructions. From step 6, gel transfer was performed to polyvinylidene difluoride membranes. For blot extraction, the membrane was divided into eight vertical slices and soaked twice in 1 ml extraction buffer (50 mM Tris-HCL, pH 9, 50 mM dithiothreitol and 0.5% Tween-20) for 2 h at room temperature with gentle shaking. The detergent was then removed using a Pierce Detergent spin column from Thermo Fisher Scientific, Inc. (cat. no. 87779), as instructed by the column manufacturer. The proteins in the extract were concentrated using Amicon Ultra 2 Centrifugal Filters with 10 kDa cut-off (cat. no. UGC501008; EMD Millipore, Billerica, MA, USA). The concentrated proteins were separated by SDS-PAGE as described previously (7).
For in-gel trypsin digestion, gel bands from the SDS PAGE were vertically cut into ten equal slices (1×1 mm). The gel pieces were dehydrated with acetonitrile (ACN) (Sigma-Aldrich; Merck KGaA), dried for 30 min and vacuumed for 10 min at 20°C, reduced in 150 µl 10 mM dithiothreitol in 100 mM ACN at 56°C for 1 h, and then alkylated with 50 mM iodoacetamide (Sigma-Aldrich; Merck KGaA) in 100 mM ACN at room temperature for 1 h in the dark. Following these washing and dehydration steps, the gel cubes were digested in 30 µl trypsin (20 ng/µl in 25 mM ACN) at 37°C overnight. The trypsin-digested peptides were subsequently extracted with 40 µl 5% trifluroacetic acid (Pierce; Thermo Fisher Scientific, Inc.) in 50% ACN for 1 h at room temperature. Finally, two rounds of drying by speed vacuum for 1 h at 45°C and resuspension in 20 µl 0.1% trifluoriacetic acid were performed, followed by MS analysis.
Digestion mixtures were loaded onto a reversed-phase fused-silica capillary emitter column [75 µm inner diameter × 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 100 Å (Thermo Fisher Scientific, Inc.)] connected to a precolumn [Acclaim PepMap 100, 75 µm × 2 cm, packed with nanoviper C18, 3 µm, 100 Å (Thermo Fisher Scientific, Inc.)]. For ultra high performance liquid chromatography (UHPLC), the column and precolumn were connected in-line to an Easy Nano LC 1000 UHPLC system (Thermo Fisher Scientific, Inc.) and were equilibrated and washed with water [Optima LC/MS Grade (W6-1) from Fisher Chemical; Thermo Fisher Scientific, Inc.]. Solvent A and solvent B were water and ACN [Fisher Chemical Optima LC/MS Grade (A955-1L); Thermo Fisher Scientific, Inc.] respectively. The peptides were gradient-eluted following application of solvent B from 2 to 98% at a flow of 350 nl/min for 1 h at 20°C, eluting 300 µl at a maximum pressure of 980 bar. Following elution, the samples were resuspended in 2% ACN, and analyzed with a Q Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Inc.). The column was fixed to a Nanospray Flex ion source from Thermo Fisher Scientific, Inc. Sheath, auxiliary and sweep gas were set to zero, and the run was in positive mode. The mass spectrometer was operated in data-dependent mode, with an automatic gain control target of 1e6 for full MS and 2e5 for data dependent-MS/MS. The isolation window was fixed to 1.3 m/z with a normalized collision energy of 28 eV. Bioinformatics analysis was performed using Thermo Proteome Discoverer v1.4 (Thermo Fisher Scientific, Inc.). Data were processed against Homo sapiens data in the Uniprot database (http://uniprot.org), allowing two missed cleavages, 10 ppm as a precursor mass tolerance and 0.02 Da for fragment mass tolerance.
MS analysis of IL6-protein complexes
Tables I and II list the proteins complexed with IL6 identified by proteomic MS analysis in C28 chondrocytes and JJ012 chondrosarcoma cells, respectively. A population of ubiquitination enzymes (<2%) were detected in C28 chondrocytes, while none were expressed in JJ012 chondrosarcoma cells. Among these enzymes were enzymes of ubiquitination machinery, namely E3 ubiquitin ligases, including E3 ubiquitin-protein ligase SH3 domain-containing ring finger 1, E3 ubiquitin-protein ligase ring finger protein 169 and E3 SUMO-protein ligase Ran-binding protein 2, as well as ubiquitin carboxyl-terminal hydrolase 33 (USP33).
Mass spectrometry analysis of proteins complexed with IL6 in human JJ012 chondrosarcoma cells.
Downregulation of IL6 expression has been observed in different tumors and is disease specific, though the cause remains unknown. Previous identification of significant downregulation of IL6 in human JJ012 chondrosarcoma cells compared with C28 chondrocytes prompted the current investigation into the mechanisms of this downregulation. In chondrosarcoma, IL6 may serve as an anti-inflammatory and anti-tumorigenic factor, based on our previous data that IL6 was downregulated by 86-fold in human chondrosarcoma compared with human C28 chondrocytes (7), indicating the possibility of a program in tumor cells with the ability to repress IL6 expression.
In the present study, a gel shift assay indicated the presence of IL6-protein complexes in C28 chondrocytes and JJ012 chondrosarcoma cells (data not shown), because we previously demonstrated (7). 2D gel electrophoresis and in-gel trypsin digestion of IL6 bands were subsequently performed to identify the IL6-protein complexes and the mechanisms involved in C28 and JJ012 cells by MS. The MS analysis detected presence of E3 ubiquitin protein ligases and USP33 hydrolase complexed with IL6 in C28 chondrocytes. Although this ligase machinery comprised a small percentage of the total proteins identified, which were overlapping between the C28 and JJ012 groups or unidentified proteins in general, this result is noteworthy, as no ubiquitination enzymes were identified in JJ012 human chondrosarcoma cells. While there is substantial data on the antitumorigenic effect of IL6 in other tumors, including undifferentiated thyroid carcinoma, thyroid cancer and bladder carcinoma (7–10,12), to the best of our knowledge, this is the first report on the potential antitumorigenic and anti-inflammatory role of IL6 in a human chondrosarcoma cell line.
It appeared inconsistent that ubiquitination machinery was prevalent in C28 cells and lacking in JJ012 cells, considering that ubiquitination generally targets proteins to proteosomal degradation, and that our previous results indicated significant downregulation of IL6 in JJ012 cells compared with C28 chondrocytes (5). However, it is possible that the absence of ubiquitination of certain unknown factors, to be determined in future studies, in JJ012 cells may lead to this downregulation of IL6.
Future in vivo experiments utilizing IL6 overexpression or knockdown in xenografts models may aid to elucidate the mechanisms of IL6 in human chondrosarcoma cells, and verify its antitumorigenic property and involvement in the process of differentiation. Following confirmation of the potential anti-proliferative function of IL6 in vivo, the next challenge will be ‘targeting the absence’ design experiments to investigate why there is downregulation of ubiquitination in chondrosarcoma, as well as the underlying mechanisms involved in this phenomena. A key approach will be to pursue studies on the posttranscriptional regulation of E3 ubiquitin ligase by miRNAs.
In conclusion, dysregulated ubiquitination may be a possible mechanism by which tumors exhibit the ability to repress IL6 expression. It is established that the microenvironments of cells, tissues and organs define gene expression. It has been demonstrated that IL6 is markedly downregulated in human chondrosarcoma cells compared with normal chondrocytes. This complies with the potential tumorigenicity and anti-inflammatory function of IL6 in chondrosarcoma. Therefore, identification of the mechanisms leading to IL6 downregulation may be important from a theoretical perspective and also for clinical practice, particularly regarding possible gene therapy applications.
The authors are thankful to Professor Sanjoy K. Bhattacharya and Ms. Maria del Carmen Piqueras from the Ophthalmology Mass Spectrometry shared instrument Core Facility of the University of Miami. The funding for mass spectrometry was provided by the National Eye Institute Ophtalmology Core Facility (grant no. P30EY14801; principal investigator Dr Vittorio Porciatti) and an unrestricted grant from Research to Prevent Blindness to the University of Miami. The study was also supported in part by the Ratcliffe Foundation of the Miami Center of Orthopedic Research and Education.
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