Investigation of mechanism of methionine aminopeptidase-2 as a molecular target against cancer-induced bone destruction

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15592111
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Surgical dentistry
• Research Institution: OKAYAMA UNIVERSITY
• Principal Investigator: SASAKI Akira OKAYAMA UNIVERSITY, GRADUATE SCHOOL OF MEDICINE, DENTISTRY, AND PHARMACEUTICAL SCIENCES, PROFESSOR, 大学院・医歯学総合研究科, 教授 (00170663)
• Co-Investigator(Kenkyū-buntansha): MESE Hiroshi OKAYAMA UNIVERSITY, GRADUATE SCHOOL OF MEDICINE, DENTISTRY, PHARMACEUTICAL SCIENCES, ASSOCIATE PROFESSOR, 大学院・医歯学総合研究科, 助教授 (40325098)
• Project Period (FY): 2003 – 2004
• Keywords: osteoclastic bone resorption / angiogenesis inhibitor / siRNA / bone invasion / bone destruction / methionine aminopeptidase-2 / transfection / microarray

Research Abstract

Osteoclastic bone resorption plays an important role on cancer-induced bone diseases like bone metastases or cancer bone invasion. We previously demonstrated that the angiogenesis inhibitor, TNP-470, inhibited not only tumor growth but also osteoclastic bone resorption. We also presented that TNP-470 has therapeutic use for bone metastasis, bone invasion and hormonal hypercalcemia in vivo models. Recently, it is reported that the candidate-target molecule of TNP-470 is methionine aminopeptidase-2 (MetAP-2), however, the relationship between TNP-470 and MetAP-2 has been still well unknown. Also, there is no information about the role of MetAP-2 on osteoclastogenesis.
In the present study, we examined the physiological function of MetAP-2 on osteoclastic bone resorption to know whether we could use MetAP-2 as a molecular target against the cancer-induced bone diseases. The expression of MetAP-2 was continuously observed as same level during the osteoclast formation and differentiation on spleen cells/RAW264.7 treated with RANKL and M-CSF. In contrast, TNP-470 markedly inhibited the osteoclast formation, but increased the expression of MetAP-2. The up-regulation of MetAP-2 may inhibit the osteoclast formation. Therefore, we transfected the expression vector with the target sequence against MetAP-2 gene into RAW264.7 using the siRNA system to decrease MetAP-2 expression. The transfectants with knock-down of MetAP-2 gene formed the large and matured osteoclasts with treatment of RANKL/M-CSF, and osteoclast number was lesser than that of control RAW cells. This indicated that the function of MetAP-2 might inhibit the osteoclast maturation. Next, we examined the effect of TNP-470 on the signal transduction of RANK/RANKL pathway. TNP-470 did not inhibits the expression of TRAF6, I-κBα and pI-κBα, but increased the transcriptional factor, NFATcl. This indicates the TNP-470 may not affect the RANK/RANKL→NF-κB pathway.