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

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 IC50 (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.