Limits...
Hsp90 Is a Novel Target Molecule of CDDO-Me in Inhibiting Proliferation of Ovarian Cancer Cells.

Qin DJ, Tang CX, Yang L, Lei H, Wei W, Wang YY, Ma CM, Gao FH, Xu HZ, Wu YL - PLoS ONE (2015)

Bottom Line: Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells.Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition.This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90.

View Article: PubMed Central - PubMed

Affiliation: Hongqiao International Institute of Medicine, Shanghai Tongren Hospital / Faculty of Basic Medicine, Chemical Biology Division of Shanghai Universities E-Institutes, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
Synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO-Me) has been shown as a promising agent against ovarian cancer. However, the underlying mechanism is not well understood. Here, we demonstrate that CDDO-Me directly interacts with Hsp90 in cells by cellular thermal shift assay. CDDO-Me treatment leads to upregulation of Hsp70 and degradation of Hsp90 clients (ErbB2 and Akt), indicating the inhibition of Hsp90 by CDDO-Me in cells. Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells. Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition. This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90. This study identifies Hsp90 as a novel target protein of CDDO-Me, and provides a novel insight into the mechanism of action of CDDO-Me in ovarian cancer cells.

No MeSH data available.


Related in: MedlinePlus

CDDO-Me interacts with Hsp90 in cells.CETSA was performed on HO8910 cells as described in materials and methods. The stabilization effect of CDDO-Me on Hsp90 and vinculin at different temperatures (A) and different doses (C) were evaluated by western blot. The intensity of the Hsp90 bands was quantified by quantity one software (B, D). Each experiment was repeated as least three times.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4489813&req=5

pone.0132337.g001: CDDO-Me interacts with Hsp90 in cells.CETSA was performed on HO8910 cells as described in materials and methods. The stabilization effect of CDDO-Me on Hsp90 and vinculin at different temperatures (A) and different doses (C) were evaluated by western blot. The intensity of the Hsp90 bands was quantified by quantity one software (B, D). Each experiment was repeated as least three times.

Mentions: In order to know whether CDDO-Me interacts with Hsp90 in cells, CETSA, a newly developed method to evaluate drug binding to target protein in cells, was performed. Ovarian cancer cells HO8910 were lysed and incubated with DMSO or CDDO-Me for half an hour at room temperature. Then, multiple aliquots of cell lysate were heated to different temperatures. After cooling, the samples were centrifugated to separate the soluble fractions from precipitated proteins and the presence of Hsp90 in the soluble fraction was examined by western blot. As shown in Fig 1A and 1B, compared with DMSO, the presence of CDDO-Me markedly increased the accumulation of Hsp90 in the soluble fraction at the temperatures examined. We also test the dose-response of CDDO-Me on Hsp90 stability to heating. As shown in Fig 1C and 1D, with the increase of CDDO-Me concentration, the accumulation of Hsp90 markedly increased. As a negative control, CDDO-Me could not increase the stability of vinculin in cells. These data suggest that CDDO-Me directly interacts with Hsp90 in cells.


Hsp90 Is a Novel Target Molecule of CDDO-Me in Inhibiting Proliferation of Ovarian Cancer Cells.

Qin DJ, Tang CX, Yang L, Lei H, Wei W, Wang YY, Ma CM, Gao FH, Xu HZ, Wu YL - PLoS ONE (2015)

CDDO-Me interacts with Hsp90 in cells.CETSA was performed on HO8910 cells as described in materials and methods. The stabilization effect of CDDO-Me on Hsp90 and vinculin at different temperatures (A) and different doses (C) were evaluated by western blot. The intensity of the Hsp90 bands was quantified by quantity one software (B, D). Each experiment was repeated as least three times.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489813&req=5

pone.0132337.g001: CDDO-Me interacts with Hsp90 in cells.CETSA was performed on HO8910 cells as described in materials and methods. The stabilization effect of CDDO-Me on Hsp90 and vinculin at different temperatures (A) and different doses (C) were evaluated by western blot. The intensity of the Hsp90 bands was quantified by quantity one software (B, D). Each experiment was repeated as least three times.
Mentions: In order to know whether CDDO-Me interacts with Hsp90 in cells, CETSA, a newly developed method to evaluate drug binding to target protein in cells, was performed. Ovarian cancer cells HO8910 were lysed and incubated with DMSO or CDDO-Me for half an hour at room temperature. Then, multiple aliquots of cell lysate were heated to different temperatures. After cooling, the samples were centrifugated to separate the soluble fractions from precipitated proteins and the presence of Hsp90 in the soluble fraction was examined by western blot. As shown in Fig 1A and 1B, compared with DMSO, the presence of CDDO-Me markedly increased the accumulation of Hsp90 in the soluble fraction at the temperatures examined. We also test the dose-response of CDDO-Me on Hsp90 stability to heating. As shown in Fig 1C and 1D, with the increase of CDDO-Me concentration, the accumulation of Hsp90 markedly increased. As a negative control, CDDO-Me could not increase the stability of vinculin in cells. These data suggest that CDDO-Me directly interacts with Hsp90 in cells.

Bottom Line: Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells.Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition.This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90.

View Article: PubMed Central - PubMed

Affiliation: Hongqiao International Institute of Medicine, Shanghai Tongren Hospital / Faculty of Basic Medicine, Chemical Biology Division of Shanghai Universities E-Institutes, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
Synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO-Me) has been shown as a promising agent against ovarian cancer. However, the underlying mechanism is not well understood. Here, we demonstrate that CDDO-Me directly interacts with Hsp90 in cells by cellular thermal shift assay. CDDO-Me treatment leads to upregulation of Hsp70 and degradation of Hsp90 clients (ErbB2 and Akt), indicating the inhibition of Hsp90 by CDDO-Me in cells. Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells. Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition. This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90. This study identifies Hsp90 as a novel target protein of CDDO-Me, and provides a novel insight into the mechanism of action of CDDO-Me in ovarian cancer cells.

No MeSH data available.


Related in: MedlinePlus