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.
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 (
Chondrosarcoma does not typically respond to conventional therapies such as chemotherapy and radiation, which necessitates the identification of novel alternative therapies (
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.
These procedures were performed as described in our recent study (
The procedures were performed according to previously described methods (
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 (
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
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 (
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 (
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 (
Future
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.
Mass spectrometry analysis of proteins complexed with IL6 in human C28 chondrocytes.
| Protein name | Accession no. |
|---|---|
| Ubiquitin carboxyl-terminal hydrolase 33 (fragment) | OS= |
| Ubiquitin-60S ribosomal protein L40 | OS= |
| Sterile alpha motif domain containing 11 splice variant ASV43 | OS= |
| Dapper homolog 1 (fragment) | OS= |
| Sarcoma antigen NY-SAR-29 (fragment) | OS= |
| TBC1 domain family member 15 (fragment) | OS= |
| cDNA FLJ61508, moderately similar to ATP-binding cassette sub-family B member 6, mitochondrial | OS= |
| Beta chimaerin isoform B2-CHNdel ex2-8,11–12 | OS= |
| 40S ribosomal protein S14 | OS= |
| Tenascin | OS= |
| Polypeptide N-acetylgalactosaminyltransferase 7 (fragment) | OS= |
| DnaJ homolog subfamily B member 11 (fragment) | OS= |
| Profilaggrin (fragment) | OS= |
| 60S ribosomal protein L27a | OS= |
| Protein FAM117B | OS= |
| Heterogeneous nuclear ribonucleoprotein K (fragment) | OS= |
| Neuropilin 2 | OS= |
| Tubulin alpha-8 chain (fragment) | OS= |
| KAT8 regulatory NSL complex subunit 1 | OS= |
| Putative EGF-like and EMI domain-containing protein 1 | OS= |
| CDNA FLJ25460 fis, clone TST09046 | OS= |
| Ribosomal protein S6 kinase alpha-5 | OS= |
| 60S ribosomal protein L7a (fragment) | OS= |
| Serine/threonine-protein kinase 35 | OS= |
| cDNA FLJ45139 fis, clone BRAWH3039623 | OS= |
| Elongation factor 1-alpha 1 | OS= |
| E3 ubiquitin-protein ligase SH3RF1 | OS= |
| Keratin, type II cytoskeletal 1 PE=1 SV=6 - Solute carrier family 45 member 4 | OS= |
| OS= |
|
| Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 | OS= |
| WD repeat-containing protein 87 | OS= |
| Prolow-density lipoprotein receptor-related protein 1 | OS= |
| 78 kDa glucose-regulated protein | OS= |
| cDNA FLJ46818 fis, clone TRACH3038399, highly similar to eukaryotic translation initiation factor 2-alpha kinase 3 | (EC 2.7.11.1) [B3KY45_HUMAN] |
| E3 SUMO-protein ligase RanBP2 | OS= |
| Interferon receptor 1 isoform 4 | OS= |
| cDNA FLJ16282 fis, clone NT2RI3005416, highly similar to F-box only protein 18 (EC 3.6.1.-) | OS= |
| Vinculin | OS= |
| Chymotrypsinogen B | OS= |
| MAP7 domain-containing protein 3 (fragment) | OS= |
| Enolase 4 | OS= |
| Myozenin-2 | OS= |
| Docking protein 6 | OS= |
| Collagen alpha-1(XIV) chain | OS= |
| Zinc finger protein 605 | OS= |
| E3 ubiquitin-protein ligase RNF169 | OS= |
| Keratin, type I cytoskeletal 9 | OS= |
| ATP synthase subunit alpha, mitochondrial | OS= |
Mass spectrometry analysis of proteins complexed with IL6 in human JJ012 chondrosarcoma cells.
| Protein name | Accession no. |
|---|---|
| Nephronectin (fragment) | OS= |
| Keratin-associated protein 19-1 | OS= |
| Ubiquitin-60S ribosomal protein L40 | OS= |
| Epithelial cell-transforming sequence 2 oncogene-like (fragment) | OS= |
| Sodium/potassium-transporting ATPase subunit beta-3 | OS= |
| TBC1 domain family member 15 (fragment) | OS= |
| cDNA FLJ61508, moderately similar to ATP-binding cassette sub-family B member 6, mitochondrial | OS= |
| ATP-binding cassette sub-family B member 8, mitochondrial | OS= |
| Heterogeneous nuclear ribonucleoprotein C-like 2 | OS= |
| Putative EGF-like and EMI domain-containing protein 1 | OS= |
| cDNA FLJ13888 fis, clone THYRO1001584 | OS= |
| Phospholysine phosphohistidine inorganic pyrophosphate phosphatase | OS= |
| Uncharacterized protein (fragment) | OS= |
| 40S ribosomal protein S14 | OS= |
| Chromosome 5 open reading frame 4, isoform CRA_a | OS= |
| 60S ribosomal protein L21 | OS= |
| S100P binding protein isoform 2 | OS= |
| cDNA FLJ26027 fis, clone PNC04328, highly similar to |
(TOMM340) [Q6ZPD_HUMAN] |
| cDNA FLJ56407, highly similar to SLIT-ROBO Rho GTPase-activating protein 2 | OS= |
| 60S ribosomal protein L27a | OS= |
| Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 | OS= |
| Neuropilin 2 | OS= |
| 60S ribosomal protein L31 | OS= |
| Protein spinster homolog 2 | OS= |
| V-type proton ATPase proteolipid subunit | OS= |
| cDNA FLJ78084, highly similar to |
OS= |
| Protein PMF1-BGLAP | OS= |
| Nuclear factor of-activated T-cells, cytoplasmic 4 (fragment) | OS= |
| Scavenger receptor class F member 1 | OS= |
| m7GpppN-mRNA hydrolase (fragment) | OS= |
| Heterogeneous nuclear ribonucleoprotein K (fragment) | OS= |
| 60S ribosomal protein L7a (fragment) | OS= |
| Elongation factor 1-alpha 1 | OS= |
| 78 kDa glucose-regulated protein | OS= |
| Zinc finger protein 90 homolog | OS= |
| Putative uncharacterized protein PCYOX1 (fragment) | OS= |
| G-protein-signaling modulator 1 | OS= |
| Vinculin | OS= |
| DNA repair protein REV1 | OS= |
| CDNA FLJ25612 fis, clone STM01228 | OS= |
| Zinc finger protein 92 homolog | OS= |
| Leucine-rich repeat-containing protein 59 | OS= |
| KN motif and ankyrin repeat domain-containing protein 2 | OS= |
| Retrotransposon-derived protein PEG10 | OS= |
| Fibrillin-1 | OS= |
| Anthrax toxin receptor 2 | OS= |
| ATP synthase subunit alpha, mitochondrial | OS= |
| ADAM9 protein | OS= |
| cDNA FLJ50932, highly similar to Zinc finger protein basonuclin-1 | OS= |
| Vigilin (fragment) | OS= |
| Alpha-protein kinase 1 | OS= |
| Neuroblast differentiation-associated protein AHNAK | OS= |
| Zinc finger protein ZFP69 | OS= |
| DNA polymerase epsilon catalytic subunit A | OS= |
| cDNA FLJ58382, highly similar to Zinc finger protein 8 | OS= |
| Chymotrypsinogen B | OS= |
| Probable ATP-dependent RNA helicase DDX46 | OS= |
| cDNA FLJ54652, highly similar to |
OS= |
| Myozenin-2 | OS= |
| Thyroid adenoma-associated protein | OS= |
| CLIP-associating protein 2 | OS= |
| Calcium/calmodulin-dependent protein kinase II alpha | OS= |
| Zinc finger protein 605 | OS= |
| Far upstream element-binding protein 2 | OS= |
| Putative uncharacterized protein XRCC5 (fragment) | OS= |
| Probable threonine-tRNA ligase 2, cytoplasmic | OS= |
| Translation initiation factor IF-2, mitochondrial | OS= |
| Dystonin | OS= |
| cDNA FLJ76304, highly similar to |
OS= |
| Polycystic kidney disease protein 1-like 1 | OS= |
| Centrosomal protein KIAA1731 | OS= |
| Ankyrin-3 | OS= |
| ATP2B2 variant protein (fragment) | OS= |