EFFECTS OF 10-HYDROXYCAMPTOTHECIN ON DIFFERENTIATION OF RAW264.7 CELLS INTO OSTEOCLASTS

Abstract

This study was designed to investigate the effect of 10-hydroxycamptothecin (10-HCPT) on osteoclastformation. RAW264.7 cells were cultured in vitro with 100 ng/ml receptor activator for nuclear factor-κB ligand (RANKL) and 30 ng/ml recombinant macrophage colony stimulating factor (M-CSF), and 10-HCPT with different solubilities were added. After five-day cultivation, tartrate-resistant acid phosphatase(TRAP) staining was used to observe the number of osteoclasts. mRNA expression of osteoclast-specificgenes, such as TRAP, cathepsin K (CTSK) and matrix metalloproteinase protease 9 (MMP-9), was detectedby real-time Polymerase Chain Reaction (PCR). The effect of 10-HCPT on the proliferation activity ofRAW264.7 cells was detected using Cell Counting Kit-8 (CCK-8). CCK-8 detection showed that 10-HCPTwith a certain concentration (1 ng/ml to 5 ng/ml) had no effect on cell proliferation (P>0.05); 10-HCPTcould inhibit the generation of osteoclasts. With the increase of the concentration of 10-HCPT, the numberof osteoclasts generated from cells cultured with 10-HCPT [1 ng/ml (86±11.14), 2 ng/ml (66.67±7.51), 5ng/ml (27.67±6.51)] was much lower than that of the control group (145±8.19), and the difference wasstatistically significant (all P=0, P<0.05); mRNA expression of osteoclast-specific gene TRAP [1 ng/ml(24.38±0.68), 2 ng/ml (20.09±1.86), 5 ng/ml (6.23±0.53)], CTSK [1 ng/ml (10.08±0.81), 2 ng/ml (7.30±0.30),5 ng/ml (3.20±0.56)] and MMP-9 [1 ng/ml (43.54±6.96), 2 ng/ml (28.28±5.83), 5 ng/ml (11.07±2.53)] wasmuch lower than that of the groups added with RANKL and M-CSF only (all P=0, P<0.05), and withthe increase of concentration of 10-HCPT, the expression of osteoclast-specific genes showed a decreasingtendency. All the findings suggest that 10-HCPT can inhibit the formation of osteoclasts by reducing theexpression of osteoclast-specific genes such as TRAP, CTSK and MMP-9.