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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 Wortmanin, SB216763 and LiCl treatment on K562 cells: (A) Uppermost panel depicts the protein level of Hsp70 by Western blot.Second panel from top depicts Hsp72 by Western blot. The third panel from top shows phosho serine9 GSK3β by Western blot. The fourth panel from top shows phospho serine473 Akt levels by Western blot. The fifth and the sixth panels from top displays Akt and actin protein levels by Western blot to denote loading controls. K-Ctrl - control K562 cells; K+0.5W- K562 cells treated with 0.5µM Wortmanin for 24h; K+1W- K562 cells treated with 1µM Wortmanin for 24h. (B) Localization of HSF1 after Wortmanin exposure. Uppermost panel represents cytosolic localization of HSF1 after 24h treatment with different concentrations of Wortmanin. The second panel from top represents actin immunoblots to indicate loading control for cytosolic fraction. The third panel from top represents nuclear localization of HSF1 after 24h treatment with different concentration of Wortmanin. The fourth panel from top represents Histone2B immunoblots to indicate loading control for nuclear fraction. K-Ctrl - control K562 cells; K+0.5W- K562 cells treated with 0.5µM Wortmanin; K+1W- K562 cells treated with 1µM Wortmanin. (C) Effect of GSK3β inhibitors on Hsp72 levels. Upper panel represents immunoblot of Hsp72 and lower panel represents actin immunoblot to indicate loading control. K-ctrl - K562 cells left untreated; K+ SB – K562 cells treated with GSK pharmacological inhibitor 5 µM SB216763 for 24h; K+ LiCl - K562 cells treated with 10mM LiCl for 24 h. All blots are representative of two separate experimental sets.
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pone-0008719-g002: Effects of Wortmanin, SB216763 and LiCl treatment on K562 cells: (A) Uppermost panel depicts the protein level of Hsp70 by Western blot.Second panel from top depicts Hsp72 by Western blot. The third panel from top shows phosho serine9 GSK3β by Western blot. The fourth panel from top shows phospho serine473 Akt levels by Western blot. The fifth and the sixth panels from top displays Akt and actin protein levels by Western blot to denote loading controls. K-Ctrl - control K562 cells; K+0.5W- K562 cells treated with 0.5µM Wortmanin for 24h; K+1W- K562 cells treated with 1µM Wortmanin for 24h. (B) Localization of HSF1 after Wortmanin exposure. Uppermost panel represents cytosolic localization of HSF1 after 24h treatment with different concentrations of Wortmanin. The second panel from top represents actin immunoblots to indicate loading control for cytosolic fraction. The third panel from top represents nuclear localization of HSF1 after 24h treatment with different concentration of Wortmanin. The fourth panel from top represents Histone2B immunoblots to indicate loading control for nuclear fraction. K-Ctrl - control K562 cells; K+0.5W- K562 cells treated with 0.5µM Wortmanin; K+1W- K562 cells treated with 1µM Wortmanin. (C) Effect of GSK3β inhibitors on Hsp72 levels. Upper panel represents immunoblot of Hsp72 and lower panel represents actin immunoblot to indicate loading control. K-ctrl - K562 cells left untreated; K+ SB – K562 cells treated with GSK pharmacological inhibitor 5 µM SB216763 for 24h; K+ LiCl - K562 cells treated with 10mM LiCl for 24 h. All blots are representative of two separate experimental sets.

Mentions: In our previous study we have conclusively established that Resveratrol downregulated Hsp70 levels and induced apoptosis in K562 cells [11]. However it was not exactly known how Resveratrol accomplished this effect on Hsp70. Exposure to Wortmanin (0.5 µM and 1µM), a potent pharmacological inhibitor for PI3K, heavily curtailed the phosphorylation of (serine 473)Akt and (serine9)Gsk3β(Fig. 2A) in K562 cells. Hsp70 levels and stress inducible form Hsp72 showed subsequent drop in protein levels on exposure to Wortmanin (Fig 2A). Since Resveratrol blocked Akt activation it was essential to demonstrate if Akt activation pathway was involved in modulating the subcellular distribution and activation of HSF1, the transcription factor responsible for expression of hsp70 gene. Measuring the levels of cytosolic and nuclear fraction of HSF1 by Western blot analysis, it was found that exposure to 0.5µM Wortmanin for 24h appreciably augmented the cytosolic localization of HSF1 which showed a further increase at 1µM concentration. In contrast Wortmanin at both the above concentrations clearly decreased the levels of nuclear HSF1 (Fig 2B). Wortmanin treatment also lowered the phosphorylated form of Gsk3β levels, which is the catalytically inactive form. This is quite understandable as Gsk3β is a substrate for phosphorylation by Akt. Therefore it was interesting to see if Gsk3β inactivation could result in upregulation of Hsp70 in K562 cells. Blocking Gsk3β by two different inhibitors LiCl (10mM) and SB216763 (5 µM) resulted in considerable increase in Hsp72 levels over untreated control after 24h of treatment (Fig 2C). LiCl exerted a stronger increase of 2.5 fold whereas SB216763 produced a moderate increase of 1.7 fold.


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 Wortmanin, SB216763 and LiCl treatment on K562 cells: (A) Uppermost panel depicts the protein level of Hsp70 by Western blot.Second panel from top depicts Hsp72 by Western blot. The third panel from top shows phosho serine9 GSK3β by Western blot. The fourth panel from top shows phospho serine473 Akt levels by Western blot. The fifth and the sixth panels from top displays Akt and actin protein levels by Western blot to denote loading controls. K-Ctrl - control K562 cells; K+0.5W- K562 cells treated with 0.5µM Wortmanin for 24h; K+1W- K562 cells treated with 1µM Wortmanin for 24h. (B) Localization of HSF1 after Wortmanin exposure. Uppermost panel represents cytosolic localization of HSF1 after 24h treatment with different concentrations of Wortmanin. The second panel from top represents actin immunoblots to indicate loading control for cytosolic fraction. The third panel from top represents nuclear localization of HSF1 after 24h treatment with different concentration of Wortmanin. The fourth panel from top represents Histone2B immunoblots to indicate loading control for nuclear fraction. K-Ctrl - control K562 cells; K+0.5W- K562 cells treated with 0.5µM Wortmanin; K+1W- K562 cells treated with 1µM Wortmanin. (C) Effect of GSK3β inhibitors on Hsp72 levels. Upper panel represents immunoblot of Hsp72 and lower panel represents actin immunoblot to indicate loading control. K-ctrl - K562 cells left untreated; K+ SB – K562 cells treated with GSK pharmacological inhibitor 5 µM SB216763 for 24h; K+ LiCl - K562 cells treated with 10mM LiCl for 24 h. All blots are representative of two separate experimental sets.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0008719-g002: Effects of Wortmanin, SB216763 and LiCl treatment on K562 cells: (A) Uppermost panel depicts the protein level of Hsp70 by Western blot.Second panel from top depicts Hsp72 by Western blot. The third panel from top shows phosho serine9 GSK3β by Western blot. The fourth panel from top shows phospho serine473 Akt levels by Western blot. The fifth and the sixth panels from top displays Akt and actin protein levels by Western blot to denote loading controls. K-Ctrl - control K562 cells; K+0.5W- K562 cells treated with 0.5µM Wortmanin for 24h; K+1W- K562 cells treated with 1µM Wortmanin for 24h. (B) Localization of HSF1 after Wortmanin exposure. Uppermost panel represents cytosolic localization of HSF1 after 24h treatment with different concentrations of Wortmanin. The second panel from top represents actin immunoblots to indicate loading control for cytosolic fraction. The third panel from top represents nuclear localization of HSF1 after 24h treatment with different concentration of Wortmanin. The fourth panel from top represents Histone2B immunoblots to indicate loading control for nuclear fraction. K-Ctrl - control K562 cells; K+0.5W- K562 cells treated with 0.5µM Wortmanin; K+1W- K562 cells treated with 1µM Wortmanin. (C) Effect of GSK3β inhibitors on Hsp72 levels. Upper panel represents immunoblot of Hsp72 and lower panel represents actin immunoblot to indicate loading control. K-ctrl - K562 cells left untreated; K+ SB – K562 cells treated with GSK pharmacological inhibitor 5 µM SB216763 for 24h; K+ LiCl - K562 cells treated with 10mM LiCl for 24 h. All blots are representative of two separate experimental sets.
Mentions: In our previous study we have conclusively established that Resveratrol downregulated Hsp70 levels and induced apoptosis in K562 cells [11]. However it was not exactly known how Resveratrol accomplished this effect on Hsp70. Exposure to Wortmanin (0.5 µM and 1µM), a potent pharmacological inhibitor for PI3K, heavily curtailed the phosphorylation of (serine 473)Akt and (serine9)Gsk3β(Fig. 2A) in K562 cells. Hsp70 levels and stress inducible form Hsp72 showed subsequent drop in protein levels on exposure to Wortmanin (Fig 2A). Since Resveratrol blocked Akt activation it was essential to demonstrate if Akt activation pathway was involved in modulating the subcellular distribution and activation of HSF1, the transcription factor responsible for expression of hsp70 gene. Measuring the levels of cytosolic and nuclear fraction of HSF1 by Western blot analysis, it was found that exposure to 0.5µM Wortmanin for 24h appreciably augmented the cytosolic localization of HSF1 which showed a further increase at 1µM concentration. In contrast Wortmanin at both the above concentrations clearly decreased the levels of nuclear HSF1 (Fig 2B). Wortmanin treatment also lowered the phosphorylated form of Gsk3β levels, which is the catalytically inactive form. This is quite understandable as Gsk3β is a substrate for phosphorylation by Akt. Therefore it was interesting to see if Gsk3β inactivation could result in upregulation of Hsp70 in K562 cells. Blocking Gsk3β by two different inhibitors LiCl (10mM) and SB216763 (5 µM) resulted in considerable increase in Hsp72 levels over untreated control after 24h of treatment (Fig 2C). LiCl exerted a stronger increase of 2.5 fold whereas SB216763 produced a moderate increase of 1.7 fold.

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