Targeted therapy for osteosarcoma using 17AAG and rapamycin: Characterization of induced apoptosis and inhibition of mTOR and AKT/MAP kinase pathways Free

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Background: Sarcomas constitute a group of diverse diseases with many subtypes and varying histologies and with adverse prognosis using current chemotherapies. Osteosarcoma is the second frequent sarcoma with low rate of response to current therapy due to inherent chemoresistance. Hence, new approaches for therapy involving targeting of specific survival pathways are needed.

Methodology: We used 17-allylamino-17-demethoxygeldanamycin (17-AAG) to block HSP90 and rapamycin to block mTOR, in 2 osteosarcoma cell lines, HOS and KHOS/NP. HOS was derived from an osteosarcoma female patient and KHOS/NP cells were derived from in vitro transfection with Kirsten murine sarcoma virus. KHOS grow on soft agar and form tumors in SCID mice. HOS and KHOS Cells were treated for 24 and 48 hours with 0-5 micromolar of 17-AAG or 0 to 10 micromolar of rapamycin. Apoptosis was determined by the extent of binding of Annexin V-FITC and cell cycle was determined by staining of cells with propidium iodide. Tetramethylrhodamine ethyl ester (TMRE) was used to detect mitochondrial membrane depolarization (MMD) and monochlorobimane (mBCl) was used to detect reduced glutathione (GSH). Stained cells were analyzed by flow cytometry (FACSAria) and quantitation was performed by the Diva software. Changes in the phosphorylation of pAkt, P70S6, Cdc2, p44Erk and pGSK were determined by Western immunoblotting (WB). Changes in the levels of HSP70, TSC1/2, activation of caspases and release of apoptosis inducing factor (AIF) from mitochondria to the cytosol were detected by WB.

Results and conclusions: 17-AAG is a good inducer of apoptosis in both sarcoma cell lines (IC50 of 50nM), involving maximal depletion of GSH and mitochondrial membrane depolarization of50 to 60%, strong activation of caspase-8 and strong translocation of AIF to the cytosol at 24 and 48 hours of treatment. 17-AAG blocked cells in mitosis (>50%) concomitant with downregulation of pCdc2, pAkt, pErk, p-mTOR and p-GSK. The effect on pErk and pAkt was observed as early as 30 and 60 minutes post treatment with 17-AAG at 24 and 48 hours of treatment. Treatment with 17-AAG resulted also in downregulation TSC2, p21, cyclin B1, cyclin D1 and upregulation of HSP70 and HSP70 C-terminal fragments, within 24 hours of treatment. No major differences were observed between HOS and KHOS cells in all parameters tested. In contrast to 17AAG, rapamycin is a weak inducer of apoptosis in sarcoma cells (IC20 at 5 micromolar), involving a small decrease in GSH and MMD (10-20%) with no translocation of AIF to the cytosol. Rapamycin slightly blocked cells in G1 concomitant with downregulation of p-p70S6, pAkt and p-mTOR and with no effect on p44Erk, pCdc2, TSC1/2 or HSPs. Concurrent treatment with both drugs resulted, at low concentrations with weak additive effect and with antagonistic effect when tested at high concentrations of both drugs.