Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.
PLoS One. 2010 Jan 14;5(1):e8719. doi: 10.1371/journal.pone.0008719.
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.
白藜芦醇能够下调慢性髓性白血病（CML）K562 细胞中 HSP70 的高内源性水平并诱导细胞凋亡。由于热休克因子 1（HSF1）控制 HSP70 的转录，我们想要探究负责 HSF1 转录激活的信号通路。
方法/主要发现：用 40μM 白藜芦醇处理细胞可迅速消除 Akt 的丝氨酸 473 磷酸化并显著降低其激酶活性。白藜芦醇阻断 Akt 通路会随后阻断 Gsk3β的丝氨酸 9 磷酸化。活性非磷酸化 Gsk3β使 HSF1 转录失活并减少 HSP70 的产生。PI3K/Akt 活性阻断剂也表现出与白藜芦醇相当的 HSP70 类似作用。用抑制剂 SB261763 或 LiCl 使 Gsk3β失活可上调 HSP70。白藜芦醇显著调节 ERK1/2 活性，表现为 T302/Y304 残基的过度磷酸化和激酶活性的同时上调。阻断 ERK1/2 激活导致 HSP70 的诱导。因此，白藜芦醇增加 ERK1/2 活性通过负调控 HSF1 活性对 HSP70 水平产生另一种负面影响。17- 烯丙基-17-脱甲氧基格尔德霉素（17AAG）是一种抑制 HSP90 伴侣并同时降解其客户蛋白 Akt 的药物，通过促进 HSF1 从细胞质向核内易位来升高 HSP70 水平。这种作用主要是由于 17AAG 抑制 Akt 和 ERK1/2 的激活。同时用白藜芦醇和 17AAG 处理 K562 细胞可使磷酸化 ERK1/2 水平接近未经处理的对照，表明它们对 ERK1/2 途径的相反作用。研究发现白藜芦醇不会干扰 K562 细胞中的 Bcr-Abl 激活。
结论/意义：因此，我们的研究全面说明了白藜芦醇在 Bcr-Abl 下游起作用，抑制 Akt 活性但刺激 ERK1/2 活性。这降低了 K562 细胞中 HSF1 的转录活性和 HSP70 的产生。此外，白藜芦醇可与化疗药物联合使用，如 HSP90 抑制剂 17AAG，它已被报道可诱导 HSP70，从而降低其化疗潜力。
