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

This study was designed to investigate the effect of 10-hydroxycamptothecin (10-HCPT) on osteoclast formation. RAW264.7 cells were cultured in vitro with 100 ng/ml receptor activator for nuclear factor-kappa B 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-specific genes, such as TRAP, cathepsin K (CTSK) and matrix metalloproteinase protease 9 (MMP-9), was detected by real-time Polymerase Chain Reaction (PCR). The effect of 10-HCPT on the proliferation activity of RAW264.7 cells was detected using Cell Counting Kit-8 (CCK-8). CCK-8 detection showed that 10-HCPT with a certain concentration (1 ng/ml to 5 ng/ml) had no effect on cell proliferation (P>0.05); 10-HCPT could inhibit the generation of osteoclasts. With the increase of the concentration of 10-HCPT, the number of osteoclasts generated from cells cultured with 10-HCPT [1 ng/ml (86 +/- 11.14), 2 ng/ml (66.67 +/- 7.51), 5 ng/ml (27.6716.51)] was much lower than that of the control group (145 +/- 8.19), and the difference was statistically 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)1, 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)] was much lower than that of the groups added with RANKL and M-CSF only (all P=0, P<0.05), and with the increase of concentration of 10-HCPT, the expression of osteoclast-specific genes showed a decreasing tendency. All the findings suggest that 10-HCPT can inhibit the formation of osteoclasts by reducing the expression of osteoclast-specific genes such as TRAP, CTSK and MMP-9.SCI(E)PubMedARTICLEwangyanwyan@126.com3789-7943