Targeted Inhibition Of MTORC1and MTORC2Signaling Effectively Inhibits Osteosarcoma

Background:Human osteosarcoma (HOS) is the most common malignant bone tumor in adolescent. The characters of occur and progress for its metastasis and high local recurrence rate, HOS is a type of cancer whose treatment requires an extensive multimodal approach including surgery, radiotherapy, and chemotherapy. Surgical amputation can achieve local tumor elimination, but20%of the osteosarcoma patients have a low grade of two years survival rate because of long distance metastasis, especially in those receiving single surgical treatment. Consequently, surgery of primary tumor combined with assistant postoperative chemotherapy has been considered significant and necessary. On using chemotherapy drugs include cisplain, epirubicin, etopside, methotrexate, and cyclophosphamide. Because of the drug resistance in osteosarcoma, only15%-65%patients can obtain acceptable effects after the completion of chemotherapy. Besides, adverse reactions caused by systematic toxicity also cannot be ignored. Cell signal target treatments to cancer are considered the new approach to research anti-tumor drug. Either blockade the energy metabolism or inducement of apoptosis of cancer cell can inhibit its occurrence and development.Objectives:Study the effect of anti-proliferation, anti-migration and pro-apoptosis in osteosarcoma in vitro by using pharmaceutical and genetic methods which inhibition of mammalian target of Rapamycin (mTOR) cell signal pathway.Methods:in vitro pharmaceutical study, observe anti-proliferation, anti-migration and pro-apoptosis effect in three kinds of osteosarcoma cells (MG63, U2OS, Saos-2) by using the first generation of mTOR signal pathway inhibitor Rapamycin and second generation mTOR dual inhibitor PP242. The anti-proliferation study included IC50assay, inhibition rate assay, colony formation assay and cell cycle assay. The anti-migration study was evaluated by wound healing assay. The pro-apoptosis study was tested by Prodium Iodide (PI) staining and apoptosis activation. Meanwhile, combination effect with cisplatin in anti-proliferation and pro-apoptosis were respectively tested with Rapamycin and PP242. In vitro genetic study, Saos-2cell was transfected by mTOR gene related small interfere RNA (siRNA) which could knockdown mTOR, Rictor, Raptor activation in cell. Anti-migration and Pro-apoptosis effect were both evaluated as described in pharmaceutical study.Results:1. mTORCl/2inhibitor PP242could effectively inhibition of proliferation in osteosarcomaPharmaceutical inhibition study showed that PP242and Rapamycin can inhibit the Phosphorylation of S6(S235/236), which is the downstream factor of mTOR complex1(mTORC1), furthermore PP242can de-regulation of the Phosphorylation of Akt (S473), the the substrate of mTOR complex2(mTORC2). Cell cycle was arrested in G1period by PP242and Rapamycin result in the significant anti-proliferation effect in colony formation assay (P<0.05) and Cell viability assay (P<0.05).2. Targeting mTORCl/2have a anti-migration effect in osteosarcomaPP242can also inhibit the migration in osteosarcoma cell line in wound healing assay, and the effect is superior to the Rapamycin (P<0.05). Also, knockdown of mTOR、 Rictor and Rapor by siRNA have the same effect as pharmacologic study.3. Traget inhibit mTORC2and (or) mTORCl/2effectively induce apoptosis in osteosarcoma.Pharmacologic inhibition of mTORC1/2by PP242showed a significantly pro-apoptosis effect in osteosarcoma cell (P<0.05), and high expression of apoptosis protein cleaved caspase-7and cleaved PARP. Genetic knockdown of mTOR, Rictor but Raptor shows dephosphorylation of akt in ser473will significantly induce apoptosis in osteosarcoma. The results demestrated that mTORC2and (or) mTORC1/2is the key role in survival effect in osteosarcoma.4. Combination treatment of PP242and siRNA increased the anti-proliferation and pro-apoptosis effect of cisplatin.Inhibition of mTORC1/2by PP242but Rapamycin combining with traditional chemotherapeutic drug cisplatin had a further potential anti-tumor effect. siRNA knockdown of Rictor and mTOR also showed the increasing effect with cisplatin.Conclusion:1. Pharmaceutical inhibition of mTORC1and mTORC2by PP242has the anti-proliferation, anti-migration and pro-apoptosis effect in osteosarcoma. Combination index demonstrated that cisplatin has the a synergistic effect with mTORC1/2inhibitor PP242.2. Genetic inhibition of mTOR1/2and mTORC2by mTOR and Rictor siRNA has show the anti-migration and pro-apoptosis effect in osteosarcoma. The result demonstrated that mTORC2probably a key role in migration and apoptosis in osteosarcoma.