Heat shock protein 90 (Hsp90) is constitutively expressed at 2-10-fold higher levels in tumor cells compared to normal cells, suggesting that it may be critically important for tumor cell growth and survival. These features make Hsp90 a potential target for anticancer drug development. Inhibition of Hsp90 activity not only results in rapid degradation of Hsp90 client proteins but also induces apoptosis of various tumor cells. Hsp90 also plays an important role in autophagy. An Hsp90 inhibitor induces autophagy through inhibition of mTOR. It is still under debate whether chemotherapy-induced autophagy in tumor cells is a protective response or is invoked to promote cell death. The aim of this study was to examine the effects of the Hsp90 inhibitor, geldanamycin (GA), on KTHOS osteosarcoma cells. We further examined whether a combination of GA and the autophagy inhibitor 3-methyl-adenine (3-MA) enhanced GA-induced apoptosis in KTHOS cells. GA had an inhibitory effect on cell proliferation and inhibited the Akt/mTOR signaling pathway in KTHOS cells. GA alone induced autophagy and apoptosis in KTHOS cells, but treatment with a combination of GA and 3-MA suppressed autophagy and induced apoptosis to a much greater extent than GA alone in these cells. It was considered that the autophagy inhibitor 3-MA suppressed a protective mechanism induced by Hsp90 inhibitor in tumor cells and induced apoptosis. Therefore, the combination of an Hsp90 inhibitor and an autophagy inhibitor may be an effective treatment for osteosarcoma because this combination effectively induces apoptotic pathways.

The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway promotes the initiation of autophagy. Although it remains under debate whether chemotherapy-induced autophagy in tumor cells is a protective response or is invoked to promote cell death, recent studies indicate that autophagy is a self-defense mechanism of cancer cells that are subjected to antitumor agents and that blocking autophagy can trigger apoptosis. The aim of this study was to examine the effects of rapamycin, an mTOR inhibitor, on MG63 osteosarcoma cells. We further examined whether the combination of rapamycin and the small molecule inhibitor of autophagy Spautin-1 (specific and potent autophagy inhibitor-1) enhanced the rapamycin-induced apoptosis in MG63 cells. We examined the effects of rapamycin treatment on cell proliferation, phosphorylation of mTOR pathway components, and autophagy by western blot analysis. Furthermore, we examined the effects of rapamycin with or without Spautin-1 on the induction of apoptosis by western blot analysis and immunohistochemical staining. We found that rapamycin inhibited cell proliferation and decreased the phosphorylation of mTOR pathway components in MG63 cells. Rapamycin induced the apoptosis of MG63 cells, and this apoptosis was enhanced by Spautin-1. It was considered that Spautin-1 suppressed the protective mechanism induced by rapamycin in tumor cells and induced apoptosis. Therefore, the combination of an mTOR inhibitor and an autophagy inhibitor may be effective in the treatment of osteosarcoma because it effectively induces the apoptotic pathway.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that functions in numerous signaling pathways initiated by diverse stimuli. The functions of GSK-3 in cancer differ depending on cell type. In the present study, we examined the effects of a specific GSK-3 inhibitor on the regulation of osteosarcoma cell proliferation and apoptosis. Immunohistochemical analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed to determine the expression pattern of GSK-3 in human osteosarcoma cells. We used the MTS assay, western blotting, measurement of single-stranded DNA and morphological analyses to study the effects of a GSK-3 inhibitor, SB216763 on osteosarcoma cell proliferation and survival. We detected an increase in mRNA expression of GSK-3 and aberrant nuclear accumulation of GSK-3 in the osteosarcoma cells. Pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of osteosarcoma cells. Inhibition of GSK-3 resulted in a decreased expression of Bcl-2 and a subsequent increase in osteosarcoma cell apoptosis via the mitochondrial pathway. The present study demonstrated that GSK-3 activity is critical for tumorigenicity and cell survival in osteosarcoma cells. Our findings suggest that GSK-3 is a potential therapeutic target for the treatment of human osteosarcoma.

The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway promotes the initiation of autophagy, and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) is well known to induce autophagy. Autophagy is a self-defense mechanism of cancer cells that are subjected to antitumor agents, and blocking autophagy can trigger apoptosis. In the present study, we demonstrate that an mTOR inhibitor, rapamycin, induces autophagy in the Nara-H malignant fibrous histiocytoma (MFH) cell line through the activation of ERK1/2. Rapamycin-induced apoptosis was enhanced following the inhibition of the MEK/ERK pathway. In the Nara-H cells, we examined the effects of rapamycin treatment on cell proliferation and on the phosphorylation of the mTOR pathway components and autophagy by western blot analysis. Furthermore, we examined the effects of rapamycin with or without the MEK inhibitor, U0126, on the induction of apoptosis by using fluorescence microscopy. Rapamycin inhibited Nara-H cell proliferation and decreased the phosphorylation of the mTOR pathway in the Nara-H cells. Rapamycin induced the apoptosis of Nara-H cells, and this apoptosis was enhanced by U0126. Simultaneously, phospho-ERK1/2 was activated by rapamycin. The present study demonstrates that rapamycin induces autophagy in Nara-H cells by activating the MEK/ERK signaling pathway, and the rapamycin-induced apoptosis can be enhanced by the MEK inhibitor, U0126. These results suggest that self-protective mechanisms involving mTOR inhibitors in Nara-H cells are prevented by the inhibition of the MEK/ERK pathway. The combination of an mTOR inhibitor (e.g., rapamycin) and an MEK inhibitor (e.g., U0126) may offer effective treatment for MFH, as this combination effectively activates apoptotic pathways.