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Hsp90 inhibitor induces autophagy and apoptosis in osteosarcoma cells.

Mori M, Hitora T, Nakamura O, Yamagami Y, Horie R, Nishimura H, Yamamoto T - Int. J. Oncol. (2014)

Bottom Line: The aim of this study was to examine the effects of the Hsp90 inhibitor, geldanamycin (GA), on KTHOS osteosarcoma cells.GA had an inhibitory effect on cell proliferation and inhibited the Akt/mTOR signaling pathway in KTHOS cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Kagawa University School of Medicine, Miki‑cho, Kagawa 761‑0793, Japan.

ABSTRACT
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‑methyladenine (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.

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The combination of geldanamycin (GA) and 3-methyladenine (3-MA) potently induces apoptosis in KTHOS cells; (A) PARP assay. Western blot analysis of PARP cleavage in KTHOS cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h. α-tubulin was used as a loading control. (B) TUNEL assay. The apoptotic rate of cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h was quantified by flow cytometry using a TUNEL assay. Values are the mean ± standard deviation (SD) of three independent experiments. (C) Annexin V-FITC/PI staining assay. The percentage of cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h was quantified using Annexin V-FITC/PI staining. Values are the mean ± SD of three independent experiments.
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f7-ijo-46-01-0047: The combination of geldanamycin (GA) and 3-methyladenine (3-MA) potently induces apoptosis in KTHOS cells; (A) PARP assay. Western blot analysis of PARP cleavage in KTHOS cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h. α-tubulin was used as a loading control. (B) TUNEL assay. The apoptotic rate of cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h was quantified by flow cytometry using a TUNEL assay. Values are the mean ± standard deviation (SD) of three independent experiments. (C) Annexin V-FITC/PI staining assay. The percentage of cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h was quantified using Annexin V-FITC/PI staining. Values are the mean ± SD of three independent experiments.

Mentions: We next determined whether GA-induced autophagy is a protective or an apoptosis-promoting mechanism. For this purpose, we assessed cellular proliferation following pre-treatment of KTHOS cells with 10 mM 3-MA, which is commonly employed as a specific inhibitor of autophagic sequestration, for 1 h prior to administration of 5 μM GA for 24 h. As shown in Fig. 6, GA inhibition of KTHOS proliferation following 3-MA pre-treatment was significantly higher than that in the absence of 3-MA treatment (P<0.05). We next used western blot analysis to examine the effect of pre-treatment with 10 mM 3-MA 1 h on the effect of GA treatment (5 μM for 24 h) on protein expression of cleaved PARP, a marker of caspase-dependent apoptosis. As shown in Fig. 7A, pre-treatment of cells with 3-MA strongly increased the cleavage of PARP induced by GA. We next examined induction of apoptotic cells by GA and the effect of 3-MA pre-treatment on such induction. Apoptotic cells were assayed by flow cytometry using the TUNEL assay. The number of apoptotic cells induced by 24 h treatment with 5 μM GA was significantly increased by 10 mM 3-MA pre-treatment (P<0.05) (Fig. 7B). We also assayed apoptotic cells using Annexin V-FITC/PI staining and fluorescence microscopy. Annexin V is a marker of early apoptosis, and PI is a marker of late apoptosis and necrosis. As shown in Fig. 7C, the number of apoptotic cells as measured by this assay that were induced by 24 h treatment with 5 μM GA was significantly increased by 10 mM 3-MA pre-treatment (P<0.001). The combined results suggest that GA induces autophagy as a protective mechanism in KTHOS cells. Furthermore, GA potently inhibits the proliferation of KTHOS cells via induction of apoptosis following 3-MA pre-treatment.


Hsp90 inhibitor induces autophagy and apoptosis in osteosarcoma cells.

Mori M, Hitora T, Nakamura O, Yamagami Y, Horie R, Nishimura H, Yamamoto T - Int. J. Oncol. (2014)

The combination of geldanamycin (GA) and 3-methyladenine (3-MA) potently induces apoptosis in KTHOS cells; (A) PARP assay. Western blot analysis of PARP cleavage in KTHOS cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h. α-tubulin was used as a loading control. (B) TUNEL assay. The apoptotic rate of cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h was quantified by flow cytometry using a TUNEL assay. Values are the mean ± standard deviation (SD) of three independent experiments. (C) Annexin V-FITC/PI staining assay. The percentage of cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h was quantified using Annexin V-FITC/PI staining. Values are the mean ± SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4238730&req=5

f7-ijo-46-01-0047: The combination of geldanamycin (GA) and 3-methyladenine (3-MA) potently induces apoptosis in KTHOS cells; (A) PARP assay. Western blot analysis of PARP cleavage in KTHOS cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h. α-tubulin was used as a loading control. (B) TUNEL assay. The apoptotic rate of cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h was quantified by flow cytometry using a TUNEL assay. Values are the mean ± standard deviation (SD) of three independent experiments. (C) Annexin V-FITC/PI staining assay. The percentage of cells pre-treated with or without 10 mM 3-MA for 1 h prior to treatment with or without 5 μM GA for 24 h was quantified using Annexin V-FITC/PI staining. Values are the mean ± SD of three independent experiments.
Mentions: We next determined whether GA-induced autophagy is a protective or an apoptosis-promoting mechanism. For this purpose, we assessed cellular proliferation following pre-treatment of KTHOS cells with 10 mM 3-MA, which is commonly employed as a specific inhibitor of autophagic sequestration, for 1 h prior to administration of 5 μM GA for 24 h. As shown in Fig. 6, GA inhibition of KTHOS proliferation following 3-MA pre-treatment was significantly higher than that in the absence of 3-MA treatment (P<0.05). We next used western blot analysis to examine the effect of pre-treatment with 10 mM 3-MA 1 h on the effect of GA treatment (5 μM for 24 h) on protein expression of cleaved PARP, a marker of caspase-dependent apoptosis. As shown in Fig. 7A, pre-treatment of cells with 3-MA strongly increased the cleavage of PARP induced by GA. We next examined induction of apoptotic cells by GA and the effect of 3-MA pre-treatment on such induction. Apoptotic cells were assayed by flow cytometry using the TUNEL assay. The number of apoptotic cells induced by 24 h treatment with 5 μM GA was significantly increased by 10 mM 3-MA pre-treatment (P<0.05) (Fig. 7B). We also assayed apoptotic cells using Annexin V-FITC/PI staining and fluorescence microscopy. Annexin V is a marker of early apoptosis, and PI is a marker of late apoptosis and necrosis. As shown in Fig. 7C, the number of apoptotic cells as measured by this assay that were induced by 24 h treatment with 5 μM GA was significantly increased by 10 mM 3-MA pre-treatment (P<0.001). The combined results suggest that GA induces autophagy as a protective mechanism in KTHOS cells. Furthermore, GA potently inhibits the proliferation of KTHOS cells via induction of apoptosis following 3-MA pre-treatment.

Bottom Line: The aim of this study was to examine the effects of the Hsp90 inhibitor, geldanamycin (GA), on KTHOS osteosarcoma cells.GA had an inhibitory effect on cell proliferation and inhibited the Akt/mTOR signaling pathway in KTHOS cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Kagawa University School of Medicine, Miki‑cho, Kagawa 761‑0793, Japan.

ABSTRACT
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‑methyladenine (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.

Show MeSH
Related in: MedlinePlus