Effect Of New Targeted Drug FIM-A On PI3K/Akt/mTOR Pathway In Osteosarcoma

Objectives1To culture rat osteosarcoma cell line UMR-106in vitro, and estabilish osteosarcomamice orthotopic model, so as to study the effects of FIM-A (AP23573),a mTORinhibitor,on osteosarcoma.2Detection of mTOR expression in rat osteoblasts and osteosarcoma cells、bonetissue of rats and mice osteosarcoma.3To Study the effect of FIM-A (AP23573) on proliferation, cell cycle, apoptosis andof the secretion on the vascular on UMR-106cell line4To explore the effect of FIM-A (AP23573) on tumor volume, PCNA, microvesseldensity and apoptosis of rat osteosarcoma.5To explore the the effect of FIM-A (AP23573) f in rat osteosarcoma and possibletherapeutic mechanism.Methods1Culturing rat osteosarcoma UMR-106cells in vitro, modeling orthotopic ratosteosarcoma model.2Using real-time quantitative PCR (Real-time PCR) method to detect the level ofmRNA of PI3K/Akt/mTOR pathway proteins expression in rat osteoblasts andosteosarcoma cells、tumor tissue and bone tissue.3Using five doses of FIM-A (AP23573) on UMR-106cell, observed the effect on cellproliferation, apoptosis, and HIF1a and VEGF secretion by way of CCK-8test, flowcytometry, Elisa (enzyme-linked immunosorbent assay).4Using three doses of FIM-A (AP23573) in orthotopic tumor model, observed theeffect on body weight of tumor-bearing nude mice, and the tumor size, growthinhibition rate, microvessel density (CD34), proliferation, PCNA antibody changes ofapoptosis factor CC3, antitumor activity evaluation. 5Observed the effect of FIM-A (AP23573) on PI3K/Akt/mTOR pathway (upstreamof mTOR,4E-BP1, phospho-4E-BP1, and downstream, p70S6K), exploring possiblemechanism in the treatment of osteosarcoma.Results1modeling rat orthotopic osteosarcoma model.2The expression of mTOR,4E-BP1, p70S6K was relatively low on rat osteoblastscells and bone tissue compared with osteosarcoma cells and tumor tissue in mice.3FIM-A (AP23573) could significantly inhibit the rat osteosarcoma UMR-106cellgrowth, the maximum concentration from10-5to10-6M; and the tumor cell cyclewere arrested in the G1phase, the secretion of HIF1α and VEGF were reduced, butfound no obvious apoptosis of cells.4FIM-A (AP23573) could significantly inhibit the growth of tumor volume In vivo.IHC showed that FIM-A (AP23573) could inhibit the proliferation of tumor cells,reduce vascular density, but no obvious effects in promoting apoptosis.5Western blot, FIM-A (AP23573) can downregulate the expression ofphospho-mTOR, phospho-4E-BP1, phospho-p70S6K on rat osteosarcoma UMR-106cells and rat osteosarcoma, so as to treat osteosarcoma.Conclusions1Modeling rat orthotopic osteosarcoma model by UMR-106cells injecting are stable.2The PI3K/Akt/mTOR pathway are abnormal and activation in osteosarcoma, mTORcan become a therapeutic target.3FIM-A (AP23573) could significantly inhibit growth of UMR-106cell in vitro; invivo experiment, FIM-A (AP23573) can effectively inhibit the growth andangiogenesis of osteosarcoma.4The effect of FIM-A (AP23573) on osteosarcoma might be related to thedown-regulation of PI3K/Akt/mTOR pathway, arresting tumor cell cycle in the G1phase, reducing the secretion of HIF1α and VEGF, thereby affecting tumorproliferation and angiogenesisa.