Effect of cytostatic proline rich polypeptide-1 on tumor suppressors of inflammation pathway signaling in chondrosarcoma

Abstract

Abstract. Cytokines produced in the tumour microenvironment exert an important role in cancer pathogenesis and in the inhibition of disease progression. Cancer of the cartilage is termed metastatic chondrosarcoma; however, the signaling events resulting in mesenchymal cell transformation to sarcoma have yet to be fully elucidated. The present study aimed to characterize the cytokine expression profile in the human JJ012 chondrosarcoma cell line, as well as the effect of cytostatic proline-rich polypeptide-1 (PRP-1). Western blot experiments demonstrated that the levels of suppressor of cytokine signaling 3 (SOCS3) were upregulated in chondrocytes compared with chondrosarcoma cells. Addition of PRP-1 restored the expression of the tumor suppressors, SOCS3 and ten-eleven-translocation methylcytosine dioxygenase 1 and 2 (TET1/2), in a dose-responsive manner. It is known that methylation of histone H3K9 was eliminated from the promoters of the inflammation-associated genes. PRP-1 inhibited H3K9 demethylase activity with an IC 50 (concentration required to give half-maximal inhibition) value of 3.72 µg/ml in the chondrosarcoma cell line. Data obtained from ELISA experiments indicated that the expression of interleukin-6 (IL-6) in chondrosarcoma cells was 86-fold lower compared with that in C28 chondrocytes. In the present study, a 53-fold downregulation of IL-6 expression in co-culture of chondrosarcoma cells and C28 chondrocytes was identified as well. Downregulation of IL-6 expression has been documented in numerous other tumor types, although the reasons for this have not been fully established. In chondrosarcoma, IL-6 manifests itself as an anti-inflammatory agent and, possibly, as an anti-tumorigenic factor. To explore protein-DNA interactions leading to such differences, a gel-shift chemiluminescent assay was performed. Gel shifts were observed for chondrosarcoma and chondrocytes in the lanes that contained nuclear cell extract and oligo-IL-6 DNA. Notably, the DNA-protein complexes in C28 chondrocytes were markedly larger compared with those in chondrosarcoma cells. The mechanisms that underpin such differences, and characterization of the interacting proteins, remain to be fully elucidated.

Analysis of IL6‑protein complexes in chondrosarcoma

“…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)(8)(9)(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).…”

“…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.…”

“…UGC501008; EMD Millipore, Billerica, MA, USA). The concentrated proteins were separated by SDS-PAGE as described previously (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)(9)(10).…”

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

“…The ability of PRP-1 to upregulate tumor suppressor miRNAs and downregulate onco-miRNAs in human chondrosarcoma JJ012 cell line was demonstrated ( 7 ). The upregulation of most tumor suppressors in chondrosarcoma ( 8 ) including inflammation related TET1/2 and SOCS3 is one of the unique PRP-1 properties, however, it depends on which molecular pathway these tumor suppressors are part of ( 9 ). PRP-1 epigenetically downregulates embryonic stem cell marker miR-302c in human chondrosarcoma and its targets Nanog, c-Myc and Bmi1 ( 10 ).…”