title: 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 osteoclast formation. RAW264.7 cells were cultured in vitro with 100 ng/ml receptor activator for nuclear factor-κ 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), 5ng/ml (27.67±6.51)] was much lower than that of the control group (145±8.19), and the difference was statistically significant (all P=0, P less than 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)] was much lower than that of the groups added with RANKL and M-CSF only (all P=0, P less than 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.
authors.affiliation: Department of Rheumatism and Immunology, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: M
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Rheumatism and Immunology, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Rheumatism and Immunology, Peking University Peoples Hospital, Beijing, China.
authors.forename: X L
authors.initials: XL
authors.lastname: Sun
authors.affiliation: Department of Rheumatism and Immunology, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: L L
authors.initials: LL
authors.lastname: Ren
authors.affiliation: Department of Rheumatism and Immunology, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: L J
authors.initials: LJ
authors.lastname: Zhang
authors.affiliation: Department of Rheumatism and Immunology, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: H Q
authors.initials: HQ
authors.lastname: Lu
mesh_terms: D000818:Animals; D018501:Antirheumatic Agents / Q000494:pharmacology*; D001172:Arthritis, Rheumatoid / Q000188:drug therapy; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D056657:Cathepsin K / Q000096:biosynthesis / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D005786:Gene Expression Regulation / Q000187:drug effects; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D010010:Osteoclasts / Q000166:cytology*; D053245:RANK Ligand / Q000494:pharmacology; D000067996:RAW 264.7 Cells / Q000166:cytology / Q000187:drug effects*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000071681:Tartrate-Resistant Acid Phosphatase / Q000096:biosynthesis / Q000235:genetics; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology*
chemical_list: D018501:Antirheumatic Agents; D053245:RANK Ligand; D012333:RNA, Messenger; D059004:Topoisomerase I Inhibitors; D016173:Macrophage Colony-Stimulating Factor; C028098:10-hydroxycamptothecin; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D056657:Cathepsin K; C534132:Ctsk protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse; D002166:Camptothecin