Chondrocyte proliferation is promoted by 7-H-pyrrolo[2,3- d]pyrimidine via up-regulation of type II collagen matrix

Purpose: To investigate the effect of 7-H-pyrrolo[2,3-d]pyrimidine derivative (7-HPPD) on the viability of chondrocytes in vitro, and to elucidate the associated mechanisms.Methods: Chondrocyte proliferation was determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay while cell cycle was assessed by flow cytometry. Western blot and immunohistochemical staining assays were used to determine protein levels.Results: The results show that 7-HPPD significantly increased the proliferation rate of chondrocytes in a concentration-dependent manner (p < 0.05). The proportion of chondrocytes in S phase increased significantly with subsequent reduction in G0/G1 phase of cell cycle on treatment with 7-HPPD (p <0.05). Thus, 7-HPPD increased the rate of chondrocyte proliferation by promoting transition through G1/S phase of cell cycle. Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis revealed that treatment of chondrocytes with 7-HPPD caused a marked increase in the level of cyclin D1, cyclin dependent kinase (CDK)-4 and CDK6 proteins (p < 0.05). Western blot and immunohistochemical staining assays showed that 7-HPPD treatment caused a significant increase in the levels of type II collagen matrix in the chondrocytes.Conclusion: Proliferation of chondrocytes is increased by 7-HPPD and this occurs by facilitating G1/S phase transition and increasing expression of cyclin D1, CDK4 and CDK6 proteins. Therefore, 7-HPPD may be developed as a chemotherapeutic agent for the treatment of osteoarthritis. Keywords: Immunohistochemistry, Type II collagen, G1/S phase, Cyclin D1, Osteoarthritis