The Effects Of MTOR Inhibition On Molecules Related To Phenotype And Growth In Osteosarcoma Cells Under Normoxia And Hypoxia

Background Basic research shows that inhibition of mammalian target of rapamycin (mTOR) inhibits the motility of osteosarcoma cells and its metastasis. However, clinical research demonstrates limited benefit from the use of mTOR inhibition. Hypoxia, one of the key features of solid tumors, may influence the activity of mTOR. mTOR servers as a nexus of various kinases. We proposed that mTOR inhibition may have complicated biological effects under normoxia and hypoxia.Purpose and methods We applied human osteosarcoma cell line MG63cells and used rapamycin to inhibit mTOR activity under normoxia and hypoxia. We investigated how mTOR inhibition would affect the expression of nucleostemin (NS), a tumor phenotype related molecule. We also examined its effect on the expressions of cAMP-dependent responsive protein (CREB) and vascular endothelial growth factor (VEGF), two molecules related to tumor cell growth and metastasis, repectively.Results Compared with human osteoblastic cell line hFOB1.19cells, MG63cells displayed higher activity of mTOR both under normoxia and hypoxia. Treatment with rapamycin at the same concentration and for the same time had similar inhibitory effects on mTOR activity in MG63cells both under normoxia and hypoxia. Under normoxia, the NS transcription and expression were increased by mTOR inhibition through enhanced nuclear translocation of Akt. Wortmannin, a specific Akt inhibitor, prevented the rapamycin-induced increases in NS expression. Hypoxia increased NS expression by enhancement of Akt activity. Since mTOR inhibition did not affect Akt activity under hypoxia, the NS expression under hypoxia was not affected by mTOR inhibition. Under normoxia, the expressions of phosphorylated CREB (pCREB) and VEGF were increased by mTOR inhibition through an extracellular-signal regulated kinase (ERK)-cAMP-dependent kinase (PKA) signaling pathway. U0126, an ERK inhibitor, prevented the increases in pCREB and VEGF. U0126, but not the PKA inhibitor H89, downregulated the relative cell proliferation rate because H89increased Akt activity. Hypoxia did not affect the expression of pCREB but increased VEGF levels. mTOR inhibition decreased the expression of pCREB. However, the mechanisms involved in the inhibitory effect exclude CREB-related kinases, calpain or the proteasome system. mTOR inhibition decreased the expression of VEGF by downregulating the expression of hypoxia-inducible factor1-a.Conclusion mTOR inhibition, a novel therapeutic method for the treatment of osteosarcoma, seems to have greater anti-tumor effects in cells under hypoxia. However, it has limited benefit as a single agent. mTOR inhibitiors should be combined with other anti-tumor agents and ERK inhibitor as well as Akt inhibitor could be taken into consideration.