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Geldanamycin-induced osteosarcoma cell death is associated with hyperacetylation and loss of mitochondrial pool of heat shock protein 60 (hsp60).

Gorska M, Marino Gammazza A, Zmijewski MA, Campanella C, Cappello F, Wasiewicz T, Kuban-Jankowska A, Daca A, Sielicka A, Popowska U, Knap N, Antoniewicz J, Wakabayashi T, Wozniak M - PLoS ONE (2013)

Bottom Line: We demonstrated that the treatment of 143B cells with geldanamycin caused a subsequent upregulation of cytoplasmic Hsp90 and Hsp70 whose activity is at least partly responsible for cancer development and drug resistance.Hyperacetylation of Hsp60 seems to be associated with anticancer activity of geldanamycin.In light of the fact that mitochondrial dysfunction plays a critical role in the apoptotic signaling pathway, the presented data may support a hypothesis that Hsp60 can be another functional part of mitochondria-related acetylome being a potential target for developing novel anticancer strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Medical University of Gdansk, Gdansk, Poland.

ABSTRACT
Osteosarcoma is one of the most malignant tumors of childhood and adolescence that is often resistant to standard chemo- and radio-therapy. Geldanamycin and geldanamycin analogs have been recently studied as potential anticancer agents for osteosarcoma treatment. Here, for the first time, we have presented novel anticancer mechanisms of geldanamycin biological activity. Moreover, we demonstrated an association between the effects of geldanamycin on the major heat shock proteins (HSPs) and the overall survival of highly metastatic human osteosarcoma 143B cells. We demonstrated that the treatment of 143B cells with geldanamycin caused a subsequent upregulation of cytoplasmic Hsp90 and Hsp70 whose activity is at least partly responsible for cancer development and drug resistance. On the other hand, geldanamycin induced upregulation of Hsp60 gene expression, and a simultaneous loss of hyperacetylated Hsp60 mitochondrial protein pool resulting in decreased viability and augmented cancer cell death. Hyperacetylation of Hsp60 seems to be associated with anticancer activity of geldanamycin. In light of the fact that mitochondrial dysfunction plays a critical role in the apoptotic signaling pathway, the presented data may support a hypothesis that Hsp60 can be another functional part of mitochondria-related acetylome being a potential target for developing novel anticancer strategies.

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GA affected HSPs protein levels in OS 143B cells.OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90, Hsp90AA1, Hsp90AB1proteins were determined by Western blotting. A. GA decreased the level of Hsp60 and induced post-translational modification of Hsp60 partially derived from the hyperacetylated isoform. B–E. GA upregulated Hsp70 (B), total cellular Hsp90 (C), Hsp90AA1 (D), Hsp90AB1 (E) protein levels. Each experiment was performed at least three times. The representative data are shown. Densitometric analysis of HSP/beta-actin was performed using Quantity one 4.5.2 software.
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pone-0071135-g003: GA affected HSPs protein levels in OS 143B cells.OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90, Hsp90AA1, Hsp90AB1proteins were determined by Western blotting. A. GA decreased the level of Hsp60 and induced post-translational modification of Hsp60 partially derived from the hyperacetylated isoform. B–E. GA upregulated Hsp70 (B), total cellular Hsp90 (C), Hsp90AA1 (D), Hsp90AB1 (E) protein levels. Each experiment was performed at least three times. The representative data are shown. Densitometric analysis of HSP/beta-actin was performed using Quantity one 4.5.2 software.

Mentions: GA is known to bind specifically to the ATP-binding site of Hsp90AA1 and Hsp90AB1 with similar affinities which results in the inhibition of Hsp90s interaction with the target proteins [18]. In order to investigate whether GA affects Hsp90 gene expression, 143B cells were treated with GA (4 µM) for 6 h. Subsequently, Hsp90AA1, Hsp90AB1, and Hsp90B1 mRNA levels were analyzed by means of Real Time PCR. Interestingly, GA caused 3- and 5-fold increase in the level of HspAB1 and Hsp90AA1 transcript, respectively (Figure 2C–D). However, GA did not significantly affect ER Hsp90B1 isoform (Figure 2E). The levels of the studied Hsp90 proteins, as observed in 143B cells after treatment with GA (4 µM) for 6 h, were also determined by Western blotting. The obtained data were in accord with those from Real Time PCR. The treatment of 143B cells with GA (4 µM) for 6 h resulted in a 1.2-, 5.5-, and 1.3-fold increase in the protein levels of the total cellular Hsp90, Hsp90AA1, and Hsp90AB1, respectively, as compared to control (Figure 3C–E). The effect of GA (4 µM) on the total cellular Hsp90 expression was then confirmed by immunofluorescence. Five random fields were acquired for each experimental condition (the representative picture is presented in Figure 4C). Cell nuclei were stained by Hoechst 33342 and shown in blue, Hsp90 immunoreactivity was shown in red. Treatment of 143B cells with GA (4 µM) remarkably increased the total cellular Hsp90 as compared to control (see Impact of GA on Hsp60 level and gene expression).


Geldanamycin-induced osteosarcoma cell death is associated with hyperacetylation and loss of mitochondrial pool of heat shock protein 60 (hsp60).

Gorska M, Marino Gammazza A, Zmijewski MA, Campanella C, Cappello F, Wasiewicz T, Kuban-Jankowska A, Daca A, Sielicka A, Popowska U, Knap N, Antoniewicz J, Wakabayashi T, Wozniak M - PLoS ONE (2013)

GA affected HSPs protein levels in OS 143B cells.OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90, Hsp90AA1, Hsp90AB1proteins were determined by Western blotting. A. GA decreased the level of Hsp60 and induced post-translational modification of Hsp60 partially derived from the hyperacetylated isoform. B–E. GA upregulated Hsp70 (B), total cellular Hsp90 (C), Hsp90AA1 (D), Hsp90AB1 (E) protein levels. Each experiment was performed at least three times. The representative data are shown. Densitometric analysis of HSP/beta-actin was performed using Quantity one 4.5.2 software.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756027&req=5

pone-0071135-g003: GA affected HSPs protein levels in OS 143B cells.OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90, Hsp90AA1, Hsp90AB1proteins were determined by Western blotting. A. GA decreased the level of Hsp60 and induced post-translational modification of Hsp60 partially derived from the hyperacetylated isoform. B–E. GA upregulated Hsp70 (B), total cellular Hsp90 (C), Hsp90AA1 (D), Hsp90AB1 (E) protein levels. Each experiment was performed at least three times. The representative data are shown. Densitometric analysis of HSP/beta-actin was performed using Quantity one 4.5.2 software.
Mentions: GA is known to bind specifically to the ATP-binding site of Hsp90AA1 and Hsp90AB1 with similar affinities which results in the inhibition of Hsp90s interaction with the target proteins [18]. In order to investigate whether GA affects Hsp90 gene expression, 143B cells were treated with GA (4 µM) for 6 h. Subsequently, Hsp90AA1, Hsp90AB1, and Hsp90B1 mRNA levels were analyzed by means of Real Time PCR. Interestingly, GA caused 3- and 5-fold increase in the level of HspAB1 and Hsp90AA1 transcript, respectively (Figure 2C–D). However, GA did not significantly affect ER Hsp90B1 isoform (Figure 2E). The levels of the studied Hsp90 proteins, as observed in 143B cells after treatment with GA (4 µM) for 6 h, were also determined by Western blotting. The obtained data were in accord with those from Real Time PCR. The treatment of 143B cells with GA (4 µM) for 6 h resulted in a 1.2-, 5.5-, and 1.3-fold increase in the protein levels of the total cellular Hsp90, Hsp90AA1, and Hsp90AB1, respectively, as compared to control (Figure 3C–E). The effect of GA (4 µM) on the total cellular Hsp90 expression was then confirmed by immunofluorescence. Five random fields were acquired for each experimental condition (the representative picture is presented in Figure 4C). Cell nuclei were stained by Hoechst 33342 and shown in blue, Hsp90 immunoreactivity was shown in red. Treatment of 143B cells with GA (4 µM) remarkably increased the total cellular Hsp90 as compared to control (see Impact of GA on Hsp60 level and gene expression).

Bottom Line: We demonstrated that the treatment of 143B cells with geldanamycin caused a subsequent upregulation of cytoplasmic Hsp90 and Hsp70 whose activity is at least partly responsible for cancer development and drug resistance.Hyperacetylation of Hsp60 seems to be associated with anticancer activity of geldanamycin.In light of the fact that mitochondrial dysfunction plays a critical role in the apoptotic signaling pathway, the presented data may support a hypothesis that Hsp60 can be another functional part of mitochondria-related acetylome being a potential target for developing novel anticancer strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Medical University of Gdansk, Gdansk, Poland.

ABSTRACT
Osteosarcoma is one of the most malignant tumors of childhood and adolescence that is often resistant to standard chemo- and radio-therapy. Geldanamycin and geldanamycin analogs have been recently studied as potential anticancer agents for osteosarcoma treatment. Here, for the first time, we have presented novel anticancer mechanisms of geldanamycin biological activity. Moreover, we demonstrated an association between the effects of geldanamycin on the major heat shock proteins (HSPs) and the overall survival of highly metastatic human osteosarcoma 143B cells. We demonstrated that the treatment of 143B cells with geldanamycin caused a subsequent upregulation of cytoplasmic Hsp90 and Hsp70 whose activity is at least partly responsible for cancer development and drug resistance. On the other hand, geldanamycin induced upregulation of Hsp60 gene expression, and a simultaneous loss of hyperacetylated Hsp60 mitochondrial protein pool resulting in decreased viability and augmented cancer cell death. Hyperacetylation of Hsp60 seems to be associated with anticancer activity of geldanamycin. In light of the fact that mitochondrial dysfunction plays a critical role in the apoptotic signaling pathway, the presented data may support a hypothesis that Hsp60 can be another functional part of mitochondria-related acetylome being a potential target for developing novel anticancer strategies.

Show MeSH
Related in: MedlinePlus