Comprehensive analysis of a new chemical compound for the treatment of osteoarthritis by proteomic approach on human chondrocytes

Osteoarthritis is a widespread and degenerative joint disorder principally characterized by progressive degradation and destruction of articular cartilage, subsequently, all structures of diarthroidal joints (subchondral bone, synovial fluid and synovial membrane) are involved. Selective cyclooxigenase (COX)-2 inhibitors were developed to prevent the gastro-intestinal adverse effects related to tNSAIDs, generally used for the treatment of OA. VA692, a new hydroxyetyl selective COX-2 inhibitor, showed anti-inflammatory, anti-nociceptive and chondroprotective properties. Proteomics is classified as a identification and characterization of the global protein content of a tissue or a particular cell type; it is a research tool for large-scale studies of structure, function and interaction of proteins. Proteomics is being applied for the study of drug mechanism of action or toxicity to identify new drugs targets. The aim of this project was to evaluate the anti-inflammatory effect of VA692, in comparison with celecoxib (already known). In addition, we would establish if the new compound present any advantages over other COX-2 inhibitors already available in therapeutic use. By iTRAQ methodology, we quantitatively analyzed the different expressed profiles in T/C-28a2 treated with the studied drugs and IL-1β. Human T/C-28a2 chondrocytes cell line were generated by Goldring group. Human articular cartilage was achieved from femoral heads of five III grade OA patients. Following an enzymatic digestion of articular cartilage, the obtained cells were incubated with VA692 and celecoxib (1μM, 0.5μM and 0.2μM) in presence of Interleukin (IL)-1β (5ng/ml) for 48 hours. The expression of inflammatory cytokines and anti-oxidant enzymes was evaluated by quantitative qRT-PCR, PGE2 release by ELISA, and apoptosis and ROS production by flow cytometry. T/C-28a2 cell line was also processed to carry out western blot analysis and employed the iTRAQ methodology. Statistical analysis was carried out by using ANOVA and Bonferroni multiple comparison tests. IL-1β-stimulated chondrocytes showed a significant increase (p<0.001) of COX-2, IL-1β, IL-6, IL-8, superoxide dismutase (SOD)-2 and catalase (CAT) gene expression, as well as of PGE2 levels in both analyzed cells type. The tested drugs significantly counteracted the effect of IL-1β, with a better modulation by VA692 1μM in T/C-28a2 cell line (p<0.01 for COX-2, IL-1β, IL-8, CAT; p<0.001 for IL-6, SOD-2 and PGE2). Regarding apoptosis and superoxide anion production, the new drug was able to significantly reduce (p<0.05) their increase induced by IL-1β (p<0.05) in T/C-28a2 cell line. Proteomic analysis identified 797 proteins in T/C-28a2 cell line, 123 of which were significantly modulated by VA692 in presence of IL-1β (p<0.001) and 34 modulated by IL-1β alone (p<0.05). 22 proteins were commonly modulated by the two groups, indicating that 101 proteins were regulated by the only effect of VA692. Among the proteins down-regulated by VA692, some with structural function were detected, responsible for cytoskeleton reorganization, as well as chaperones (heat shock proteins) and glycolitic enzymes. Proteins involved in calcium metabolism and in ribosome biogenesis resulted up-regulated instead, as well as Cu-Zn SOD, another member of SOD family, as confirmed by western blot analysis on SOD-2. Our data underlined the anti-inflammatory effect of VA692, suggesting also its anti-apoptotic and anti-oxidant role. The proteomic profile demonstrated that VA692 induced not only an anti-inflammatory regulation in chondrocytes but, interestingly, seemed to regulate their anabolic response. On the basis of our results, we can affirm that VA692 has more beneficial effect in comparison to celecoxib in particular regarding the modulation of oxidant/anti-oxidant system and of the proteome profile. So, we hypothesize that VA692 could present any advantages over other COX-2 inhibitors already available for therapeutic use. However, further in vitro and in vivo experiments are necessary to elucidate and confirm the importance of this pharmacological compound before to in therapeutic approach of joint diseases.