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Modulation of Akt and ERK1/2 pathways by resveratrol in chronic myelogenous leukemia (CML) cells results in the downregulation of Hsp70.

Banerjee Mustafi S, Chakraborty PK, Raha S - PLoS ONE (2010)

Bottom Line: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity.Blocking ERK1/2 activation resulted in induction of Hsp70.Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.

View Article: PubMed Central - PubMed

Affiliation: Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

ABSTRACT

Background: Resveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70) in Chronic Myelogenous Leukemia (CML) K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1) controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1.

Methodology/principal findings: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3beta. Active non-phosphorylated Gsk3beta rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3beta activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG), a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.

Conclusion/significance: Thus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK1/2 activity. This brings down the transcriptional activity of HSF1 and Hsp70 production in K562 cells. Additionally, Resveratrol can be used in combination with chemotherapeutic agents such as 17AAG, an Hsp90 inhibitor reported to induce Hsp70 and hence compromise its chemotherapeutic potential.

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Related in: MedlinePlus

Alteration in Akt activation in response to Resveratrol: (A) Akt phosphorylation shown in immunoblot.In the upper panel phospho serine473 Akt immunoblot is displayed. In the middle panel Akt and in the lower panel Actin immunoblots are displayed to indicate loading controls. K-Ctrl – untreated K562 cells; K+ R1H- K562 cells treated with Resveratrol for 1h; K+ R3H- K562 cells treated with Resveratrol for 3h; K+ R6H- K562 cells treated with Resveratrol for 6h; K+ R9H- K562 cells treated with Resveratrol for 9h. (B) The panel displays the ratio (mean±SD) of the P-Akt protein/Akt protein band densities of K562 control cells (K-Ctrl) and 6h Resveratrol treated cells (K+ R6H) from three experiments. (C) Results of Akt Kinase assay are displayed as immunoblot. Total Akt was immunoprecipitated from Resveratrol treated or untreated cell lysates (containing equal amount of total protein) and kinase assay was performed with equal amount of the precipitated beads and GSK3β synthetic peptide and ATP as substrates and P- GSK3β was detected by Western Blot using anti serine9 GSK3β antibody. K-Ctrl – untreated K562 cells; K+ Res (6H) - K562 cells treated with Resveratrol for 6 h. (D) Cellular localization of phospho serine473 Akt by fluorescence microscopy. A: Untreated K562 cells (K-Ctrl) processed for visualization of P-Akt with secondary antibody FITC conjugate.; B: Untreated K562 cells processed for visualization of DNA using specific stain DAPI; C-: DIC images of the cells; D: K562 cells treated with Resveratrol for 6 h and processed for visualization of P-Akt with secondary antibody FITC conjugate.; .E: K562 cells treated with Resveratrol for 6 h and processed for visualization of DNA using specific stain DAPI; F: DIC images of the cells. (E) Phospho-GSK3 immunoblot. Upper panel shows phospho-GSK3β and lower panel shows actin as loading control. K-Ctrl – untreated K562 cells; K+ R12H - K562 cells treated with Resveratrol for 12h; K+ R24H - K562 cells treated with Resveratrol for 24h. The bar represents 10µm. All the blots and micrographs are representative of two to three sets of separate experiments. Concentration of Resveratrol is 40µM. Details of the experiments are described in section-2.
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pone-0008719-g001: Alteration in Akt activation in response to Resveratrol: (A) Akt phosphorylation shown in immunoblot.In the upper panel phospho serine473 Akt immunoblot is displayed. In the middle panel Akt and in the lower panel Actin immunoblots are displayed to indicate loading controls. K-Ctrl – untreated K562 cells; K+ R1H- K562 cells treated with Resveratrol for 1h; K+ R3H- K562 cells treated with Resveratrol for 3h; K+ R6H- K562 cells treated with Resveratrol for 6h; K+ R9H- K562 cells treated with Resveratrol for 9h. (B) The panel displays the ratio (mean±SD) of the P-Akt protein/Akt protein band densities of K562 control cells (K-Ctrl) and 6h Resveratrol treated cells (K+ R6H) from three experiments. (C) Results of Akt Kinase assay are displayed as immunoblot. Total Akt was immunoprecipitated from Resveratrol treated or untreated cell lysates (containing equal amount of total protein) and kinase assay was performed with equal amount of the precipitated beads and GSK3β synthetic peptide and ATP as substrates and P- GSK3β was detected by Western Blot using anti serine9 GSK3β antibody. K-Ctrl – untreated K562 cells; K+ Res (6H) - K562 cells treated with Resveratrol for 6 h. (D) Cellular localization of phospho serine473 Akt by fluorescence microscopy. A: Untreated K562 cells (K-Ctrl) processed for visualization of P-Akt with secondary antibody FITC conjugate.; B: Untreated K562 cells processed for visualization of DNA using specific stain DAPI; C-: DIC images of the cells; D: K562 cells treated with Resveratrol for 6 h and processed for visualization of P-Akt with secondary antibody FITC conjugate.; .E: K562 cells treated with Resveratrol for 6 h and processed for visualization of DNA using specific stain DAPI; F: DIC images of the cells. (E) Phospho-GSK3 immunoblot. Upper panel shows phospho-GSK3β and lower panel shows actin as loading control. K-Ctrl – untreated K562 cells; K+ R12H - K562 cells treated with Resveratrol for 12h; K+ R24H - K562 cells treated with Resveratrol for 24h. The bar represents 10µm. All the blots and micrographs are representative of two to three sets of separate experiments. Concentration of Resveratrol is 40µM. Details of the experiments are described in section-2.

Mentions: We first confirmed if Resveratrol has any significant role in altering the Akt activity. 40µM Resveratrol significantly curtailed the phosphorylation (Ser 473) of Akt by >25% within 1h of treatment of K562 cells (Fig. 1A). This effect was further bolstered at later time points up to 9h with a significant drop of around 10 fold (P<0.05) registered at 6h (Fig 1B). Phosphorylation of Akt at 473 serine residue is directly linked to the kinase activity of the protein. Therefore a kinase assay was performed to determine if Resveratrol could also efficiently alter the catalytic activity of Akt. Exposure to 40µM Resveratrol for 6 h resulted in a significant drop (84%±7%) in the kinase activity of Akt (Fig. 1C). Resveratrol thus effectively compromised the phosphorylation and activity of one of the major oncoprotein, Akt, highly active in chronic myelogenous leukemia. Fig. 1D demonstrates that phosphorylated Akt is predominantly located in the nucleus of K562 cells but in K562 cells treated with Resveratrol for 6h phosphorylated Akt could be visualized neither in the nucleus nor in other parts of the cell. Akt in both cases (untreated control and Resveratrol treated) is almost equally distributed in the cytoplasm and nucleus (Fig. S1). Fig. 1E shows the phosphorylation status of the Akt substrate GSK-3β (serine9) which is markedly phosphorylated in K562 cells but the phosphorylation is diminished drastically upon Resveratrol exposure.


Modulation of Akt and ERK1/2 pathways by resveratrol in chronic myelogenous leukemia (CML) cells results in the downregulation of Hsp70.

Banerjee Mustafi S, Chakraborty PK, Raha S - PLoS ONE (2010)

Alteration in Akt activation in response to Resveratrol: (A) Akt phosphorylation shown in immunoblot.In the upper panel phospho serine473 Akt immunoblot is displayed. In the middle panel Akt and in the lower panel Actin immunoblots are displayed to indicate loading controls. K-Ctrl – untreated K562 cells; K+ R1H- K562 cells treated with Resveratrol for 1h; K+ R3H- K562 cells treated with Resveratrol for 3h; K+ R6H- K562 cells treated with Resveratrol for 6h; K+ R9H- K562 cells treated with Resveratrol for 9h. (B) The panel displays the ratio (mean±SD) of the P-Akt protein/Akt protein band densities of K562 control cells (K-Ctrl) and 6h Resveratrol treated cells (K+ R6H) from three experiments. (C) Results of Akt Kinase assay are displayed as immunoblot. Total Akt was immunoprecipitated from Resveratrol treated or untreated cell lysates (containing equal amount of total protein) and kinase assay was performed with equal amount of the precipitated beads and GSK3β synthetic peptide and ATP as substrates and P- GSK3β was detected by Western Blot using anti serine9 GSK3β antibody. K-Ctrl – untreated K562 cells; K+ Res (6H) - K562 cells treated with Resveratrol for 6 h. (D) Cellular localization of phospho serine473 Akt by fluorescence microscopy. A: Untreated K562 cells (K-Ctrl) processed for visualization of P-Akt with secondary antibody FITC conjugate.; B: Untreated K562 cells processed for visualization of DNA using specific stain DAPI; C-: DIC images of the cells; D: K562 cells treated with Resveratrol for 6 h and processed for visualization of P-Akt with secondary antibody FITC conjugate.; .E: K562 cells treated with Resveratrol for 6 h and processed for visualization of DNA using specific stain DAPI; F: DIC images of the cells. (E) Phospho-GSK3 immunoblot. Upper panel shows phospho-GSK3β and lower panel shows actin as loading control. K-Ctrl – untreated K562 cells; K+ R12H - K562 cells treated with Resveratrol for 12h; K+ R24H - K562 cells treated with Resveratrol for 24h. The bar represents 10µm. All the blots and micrographs are representative of two to three sets of separate experiments. Concentration of Resveratrol is 40µM. Details of the experiments are described in section-2.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806839&req=5

pone-0008719-g001: Alteration in Akt activation in response to Resveratrol: (A) Akt phosphorylation shown in immunoblot.In the upper panel phospho serine473 Akt immunoblot is displayed. In the middle panel Akt and in the lower panel Actin immunoblots are displayed to indicate loading controls. K-Ctrl – untreated K562 cells; K+ R1H- K562 cells treated with Resveratrol for 1h; K+ R3H- K562 cells treated with Resveratrol for 3h; K+ R6H- K562 cells treated with Resveratrol for 6h; K+ R9H- K562 cells treated with Resveratrol for 9h. (B) The panel displays the ratio (mean±SD) of the P-Akt protein/Akt protein band densities of K562 control cells (K-Ctrl) and 6h Resveratrol treated cells (K+ R6H) from three experiments. (C) Results of Akt Kinase assay are displayed as immunoblot. Total Akt was immunoprecipitated from Resveratrol treated or untreated cell lysates (containing equal amount of total protein) and kinase assay was performed with equal amount of the precipitated beads and GSK3β synthetic peptide and ATP as substrates and P- GSK3β was detected by Western Blot using anti serine9 GSK3β antibody. K-Ctrl – untreated K562 cells; K+ Res (6H) - K562 cells treated with Resveratrol for 6 h. (D) Cellular localization of phospho serine473 Akt by fluorescence microscopy. A: Untreated K562 cells (K-Ctrl) processed for visualization of P-Akt with secondary antibody FITC conjugate.; B: Untreated K562 cells processed for visualization of DNA using specific stain DAPI; C-: DIC images of the cells; D: K562 cells treated with Resveratrol for 6 h and processed for visualization of P-Akt with secondary antibody FITC conjugate.; .E: K562 cells treated with Resveratrol for 6 h and processed for visualization of DNA using specific stain DAPI; F: DIC images of the cells. (E) Phospho-GSK3 immunoblot. Upper panel shows phospho-GSK3β and lower panel shows actin as loading control. K-Ctrl – untreated K562 cells; K+ R12H - K562 cells treated with Resveratrol for 12h; K+ R24H - K562 cells treated with Resveratrol for 24h. The bar represents 10µm. All the blots and micrographs are representative of two to three sets of separate experiments. Concentration of Resveratrol is 40µM. Details of the experiments are described in section-2.
Mentions: We first confirmed if Resveratrol has any significant role in altering the Akt activity. 40µM Resveratrol significantly curtailed the phosphorylation (Ser 473) of Akt by >25% within 1h of treatment of K562 cells (Fig. 1A). This effect was further bolstered at later time points up to 9h with a significant drop of around 10 fold (P<0.05) registered at 6h (Fig 1B). Phosphorylation of Akt at 473 serine residue is directly linked to the kinase activity of the protein. Therefore a kinase assay was performed to determine if Resveratrol could also efficiently alter the catalytic activity of Akt. Exposure to 40µM Resveratrol for 6 h resulted in a significant drop (84%±7%) in the kinase activity of Akt (Fig. 1C). Resveratrol thus effectively compromised the phosphorylation and activity of one of the major oncoprotein, Akt, highly active in chronic myelogenous leukemia. Fig. 1D demonstrates that phosphorylated Akt is predominantly located in the nucleus of K562 cells but in K562 cells treated with Resveratrol for 6h phosphorylated Akt could be visualized neither in the nucleus nor in other parts of the cell. Akt in both cases (untreated control and Resveratrol treated) is almost equally distributed in the cytoplasm and nucleus (Fig. S1). Fig. 1E shows the phosphorylation status of the Akt substrate GSK-3β (serine9) which is markedly phosphorylated in K562 cells but the phosphorylation is diminished drastically upon Resveratrol exposure.

Bottom Line: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity.Blocking ERK1/2 activation resulted in induction of Hsp70.Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.

View Article: PubMed Central - PubMed

Affiliation: Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

ABSTRACT

Background: Resveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70) in Chronic Myelogenous Leukemia (CML) K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1) controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1.

Methodology/principal findings: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3beta. Active non-phosphorylated Gsk3beta rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3beta activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG), a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.

Conclusion/significance: Thus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK1/2 activity. This brings down the transcriptional activity of HSF1 and Hsp70 production in K562 cells. Additionally, Resveratrol can be used in combination with chemotherapeutic agents such as 17AAG, an Hsp90 inhibitor reported to induce Hsp70 and hence compromise its chemotherapeutic potential.

Show MeSH
Related in: MedlinePlus