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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

Effects of 17AAG and Resveratrol treatment on K562 cells: (A) Effects of 17-AAG on phosphorylations of Akt and ERK 1/2, and protein levels of Hsp70.Uppermost panel depicts phospho serine473 Akt levels by Western blot. Second panel from top depicts Akt by Western blot to represent protein loading. The third panel from top indicates phospho threonine202/phospho tyrosine204 p42/44 MAPK immunoblot. The fourth panel from top shows p42/44 MAPK by Western blot to represent corresponding loading control. The fifth panel from top displays Hsp70 protein levels and sixth panel depicts actin protein levels by Western blot. K-Ctrl - control K562 cells; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+5G- K562 cells treated with 5µM 17AAG for 24h. (B) Effect of 17-AAG on GSK3β. The upper panel shows immunoblots of phospho serine9 GSK3β in response to treatment with different dose concentration of 17AAG for 24h. The lower panel represents actin immunoblots. K-Ctrl - control K562 cells; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+5G- K562 cells treated with 5µM 17AAG for 24h. (C) Effects of a combination of 17-AAG and Resveratrol on phosphorylation status of ERK1/2. The top panel indicates phospho threonine202/phospho tyrosine204 p42/44 ERK immunoblot and the bottom panel indicates p42/44 ERK to represent protein loading.. K-Ctrl - untreated K562 cells; K+ 40Res - K562 cells treated with 40µM Resveratrol for 24h; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+2.5G+40Res - K562 cells treated simultaneously with 2.5µM 17AAG and 40µM Resveratrol for 24h. All blots are representative of two to three independent experimental sets.
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pone-0008719-g004: Effects of 17AAG and Resveratrol treatment on K562 cells: (A) Effects of 17-AAG on phosphorylations of Akt and ERK 1/2, and protein levels of Hsp70.Uppermost panel depicts phospho serine473 Akt levels by Western blot. Second panel from top depicts Akt by Western blot to represent protein loading. The third panel from top indicates phospho threonine202/phospho tyrosine204 p42/44 MAPK immunoblot. The fourth panel from top shows p42/44 MAPK by Western blot to represent corresponding loading control. The fifth panel from top displays Hsp70 protein levels and sixth panel depicts actin protein levels by Western blot. K-Ctrl - control K562 cells; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+5G- K562 cells treated with 5µM 17AAG for 24h. (B) Effect of 17-AAG on GSK3β. The upper panel shows immunoblots of phospho serine9 GSK3β in response to treatment with different dose concentration of 17AAG for 24h. The lower panel represents actin immunoblots. K-Ctrl - control K562 cells; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+5G- K562 cells treated with 5µM 17AAG for 24h. (C) Effects of a combination of 17-AAG and Resveratrol on phosphorylation status of ERK1/2. The top panel indicates phospho threonine202/phospho tyrosine204 p42/44 ERK immunoblot and the bottom panel indicates p42/44 ERK to represent protein loading.. K-Ctrl - untreated K562 cells; K+ 40Res - K562 cells treated with 40µM Resveratrol for 24h; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+2.5G+40Res - K562 cells treated simultaneously with 2.5µM 17AAG and 40µM Resveratrol for 24h. All blots are representative of two to three independent experimental sets.

Mentions: The findings above indicated the important roles of Akt and ERK1/2 activation in down regulation of Hsp70. However the question that needs to be addressed is how diminishing Akt activation by17AAG treatment (Fig 4A) could simultaneously result in induction of stress response by sharply augmenting the Hsp70 levels (Fig 4A). 17AAG blocks the chaperoning activity of Hsp90, prompting degradation of its client protein Akt. This is well demonstrated by the dose dependent decline of Akt and phospho serine 473 Akt levels in K562 cells treated with 2.5µM and 5 µM concentration of 17AAG (Fig 4A). Phospho Gsk3β levels also showed a sharp decline with treatment with 2.5 µM 17AAG and this de-phosphorylation of Gsk3β was further augmented at higher concentration of 5 µM 17AAG (Fig 4B). Since down regulation of Akt activity and corresponding Gsk3β activation both are known to be associated with Hsp70 down regulation (Fig 1,2) the effect of 17AAG on ERK1/2 gains importance and holds the key to this apparent puzzle. Exposure to 17AAG induced a distinct de-phosphorylation of ERK1/2 (T-202/Y-204) by several fold (8 fold, P<0.05). When combined with Resveratrol, 17AAG demonstrated an opposite effect on ERK1/2 activation in K562 cells. After simultaneous exposure of K562 to both 40 µM Resveratrol and 2.5 µM 17AAG for 24h the levels of P-ERK1/2 were much higher than that in cells treated with 17-AAG alone and were close to untreated controls (Fig 4C). In comparison, ERK1/2 Phosphorylation status in response to only 40 µM Resveratrol recorded almost 2 fold increase over control (Fig 4C).


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)

Effects of 17AAG and Resveratrol treatment on K562 cells: (A) Effects of 17-AAG on phosphorylations of Akt and ERK 1/2, and protein levels of Hsp70.Uppermost panel depicts phospho serine473 Akt levels by Western blot. Second panel from top depicts Akt by Western blot to represent protein loading. The third panel from top indicates phospho threonine202/phospho tyrosine204 p42/44 MAPK immunoblot. The fourth panel from top shows p42/44 MAPK by Western blot to represent corresponding loading control. The fifth panel from top displays Hsp70 protein levels and sixth panel depicts actin protein levels by Western blot. K-Ctrl - control K562 cells; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+5G- K562 cells treated with 5µM 17AAG for 24h. (B) Effect of 17-AAG on GSK3β. The upper panel shows immunoblots of phospho serine9 GSK3β in response to treatment with different dose concentration of 17AAG for 24h. The lower panel represents actin immunoblots. K-Ctrl - control K562 cells; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+5G- K562 cells treated with 5µM 17AAG for 24h. (C) Effects of a combination of 17-AAG and Resveratrol on phosphorylation status of ERK1/2. The top panel indicates phospho threonine202/phospho tyrosine204 p42/44 ERK immunoblot and the bottom panel indicates p42/44 ERK to represent protein loading.. K-Ctrl - untreated K562 cells; K+ 40Res - K562 cells treated with 40µM Resveratrol for 24h; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+2.5G+40Res - K562 cells treated simultaneously with 2.5µM 17AAG and 40µM Resveratrol for 24h. All blots are representative of two to three independent experimental sets.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0008719-g004: Effects of 17AAG and Resveratrol treatment on K562 cells: (A) Effects of 17-AAG on phosphorylations of Akt and ERK 1/2, and protein levels of Hsp70.Uppermost panel depicts phospho serine473 Akt levels by Western blot. Second panel from top depicts Akt by Western blot to represent protein loading. The third panel from top indicates phospho threonine202/phospho tyrosine204 p42/44 MAPK immunoblot. The fourth panel from top shows p42/44 MAPK by Western blot to represent corresponding loading control. The fifth panel from top displays Hsp70 protein levels and sixth panel depicts actin protein levels by Western blot. K-Ctrl - control K562 cells; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+5G- K562 cells treated with 5µM 17AAG for 24h. (B) Effect of 17-AAG on GSK3β. The upper panel shows immunoblots of phospho serine9 GSK3β in response to treatment with different dose concentration of 17AAG for 24h. The lower panel represents actin immunoblots. K-Ctrl - control K562 cells; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+5G- K562 cells treated with 5µM 17AAG for 24h. (C) Effects of a combination of 17-AAG and Resveratrol on phosphorylation status of ERK1/2. The top panel indicates phospho threonine202/phospho tyrosine204 p42/44 ERK immunoblot and the bottom panel indicates p42/44 ERK to represent protein loading.. K-Ctrl - untreated K562 cells; K+ 40Res - K562 cells treated with 40µM Resveratrol for 24h; K+2.5G- K562 cells treated with 2.5µM 17AAG for 24h; K+2.5G+40Res - K562 cells treated simultaneously with 2.5µM 17AAG and 40µM Resveratrol for 24h. All blots are representative of two to three independent experimental sets.
Mentions: The findings above indicated the important roles of Akt and ERK1/2 activation in down regulation of Hsp70. However the question that needs to be addressed is how diminishing Akt activation by17AAG treatment (Fig 4A) could simultaneously result in induction of stress response by sharply augmenting the Hsp70 levels (Fig 4A). 17AAG blocks the chaperoning activity of Hsp90, prompting degradation of its client protein Akt. This is well demonstrated by the dose dependent decline of Akt and phospho serine 473 Akt levels in K562 cells treated with 2.5µM and 5 µM concentration of 17AAG (Fig 4A). Phospho Gsk3β levels also showed a sharp decline with treatment with 2.5 µM 17AAG and this de-phosphorylation of Gsk3β was further augmented at higher concentration of 5 µM 17AAG (Fig 4B). Since down regulation of Akt activity and corresponding Gsk3β activation both are known to be associated with Hsp70 down regulation (Fig 1,2) the effect of 17AAG on ERK1/2 gains importance and holds the key to this apparent puzzle. Exposure to 17AAG induced a distinct de-phosphorylation of ERK1/2 (T-202/Y-204) by several fold (8 fold, P<0.05). When combined with Resveratrol, 17AAG demonstrated an opposite effect on ERK1/2 activation in K562 cells. After simultaneous exposure of K562 to both 40 µM Resveratrol and 2.5 µM 17AAG for 24h the levels of P-ERK1/2 were much higher than that in cells treated with 17-AAG alone and were close to untreated controls (Fig 4C). In comparison, ERK1/2 Phosphorylation status in response to only 40 µM Resveratrol recorded almost 2 fold increase over control (Fig 4C).

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