Modulation of Akt and ERK1/2 Pathways by Resveratrol in Chronic Myelogenous Leukemia (CML) Cells Results in the Downregulation of Hsp70

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

Mechanisms of plant adaptation/memory in rice seedlings under arsenic and heat stress: expression of heat-shock protein gene HSP70

Imprints of stress response by rice seedlings in terms of expression levels of stress response gene HSP70 are characterised . The response to arsenic and/or heat shock are shown to be additive for the same stress or the combined stresses, indicating a commonality of signalling pathways

EhMAPK, the Mitogen-Activated Protein Kinase from Entamoeba histolytica Is Associated with Cell Survival

Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H2O2 treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H2O2 treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H2O2 treatment reduced parasite viability significantly but heat shock and 0.5 mM H2O2 treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified

Differential alterations of positive and negative regulators of beta catenin enhance endogenous expression and activity of beta catenin in A549 non small cell lung cancer (NSCLC) cells

Beta catenin has been well documented in previous studies to be involved in non small cell lung cancer (NSCLC). Beta catenin abundance and transcriptional activity are significantly regulated by several factors. Though it is well known that Akt and Gsk3 beta are respective positive and negative regulators of beta catenin, however, no single study has so far documented how the expression and activity of both positive as well as negative regulators play favorable role on beta catenin expression and activity in NSCLC. In this study, we compared expression and activity of beta catenin and its regulators in normal lung cell WI38 and NSCLC cell A549 by western blot, qRT-PCR and luciferase assay. We observed that beta catenin positive regulators (Akt and Hsp90) and negative regulators (Gsk3 beta and microRNA-214) have differential expression and/or activity in NSCLC cell A549. However the differentially altered statuses of both the positive and negative regulators rendered cumulative positive effect on beta catenin expression and activity in A549. Our study thus suggests that chemotherapeutic modulations of regulating factors are crucial when abrogation and/or inhibition of key oncogenic proteins are necessary for cancer chemotherapy

<span style="font-size:14.0pt;font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-fareast-language: EN-US;mso-bidi-language:AR-SA">Purification and characterization of receptors for myoinositol trisphosphate and myoinositol tetrakis phosphate from <i>Entamoeba histolytica</i></span>

253-257The microsomal fraction from the log phase of Entamoeba histolytica cells contains Ins(1,4,5)P3 and Ins(1 ,3,4,5)P4 binding activity. The binding proteins/receptors for both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 were purified and found to be specific for each ligand. The molecular masses for native proteins for InsP3 and Ins P4 are 138 kDa and 130 kDa respectively having subunits of 69 kDa and 64 kDa respectively. That these proteins are associated with Ca2+ release was confirmed by including these proteins separately in proteoliposomes and adding Ins P3 and Ins P4 in both the cases

Benzo(a)pyrene induced p53 mediated male germ cell apoptosis: Synergistic protective effects of curcumin and resveratrol

B(a)P (Benzo(a)pyrene) is an environmental toxicant that induces male germ cell apoptosis. Curcumin and resveratrol are phytochemicals with cytoprotective and anti-oxidative properties. At the same time resveratrol is also a natural Aryl hydrocarbon Receptor (AhR) antagonist. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted the synergistic protective effect of curcumin and resveratrol against B(a)P induced p53 mediated germ cell apoptosis. Curcumin-resveratrol significantly prevented B(a)P induced decrease in sperm cell count and motility, as well as increased serum testosterone level. Curcumin-resveratrol co-treatment actively protected B(a)P induced testicular germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3, 8 and 9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2 and Apaf1. B(a)P induced testicular ROS (Reactive Oxygen Species) generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1(Cytochrome P4501A1) expression. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis

Beta catenin is degraded by both caspase-3 and proteasomal activity during resveratrol-induced apoptosis in HeLa cells in a GSK3β-independent manner

7-13Increased activity of β-catenin, an important transcriptional activator for survival and proliferation-associated genes has been linked with many cancers. We examined whether β-catenin is a target of resveratrol and whether its degradation contributes to the pro-apoptotic effects of resveratrol. HeLa cells were exposed to 60 µM resveratrol for 48 h. Apoptosis was confirmed by measurement of annexin V externalization, caspase-3 activation and DNA fragmentation. Induction of apoptosis was observed as early as 12 h, when both caspase-3 activation and PARP (poly ADP ribose polymerase) cleavage occurred. Nuclear β-catenin levels remained unchanged for 48 h during resveratrol exposure. However, extranuclear cell lysate β-catenin underwent a decrease at a late stage of apoptosis namely at 36-48 h. Alterations in the phosphorylation status of Akt/GSK3β were not observed during resveratrol-induced apoptosis. Furthermore, inhibition of GSK3β activity which is largely responsible for β-catenin degradation failed to influence β-catenin stability. However, inhibition of caspase-3 activity prevented the decline in β-catenin levels at 36-48 h of resveratrol exposure. Lactacystin, a proteosomal inhibitor also prevented the degradation of β-catenin by resveratrol. In conclusion, resveratrol induced apoptosis in HeLa cells in an Akt/GSK3β-independent manner and down-regulated β-catenin levels during apoptosis through action of caspase-3 and proteasomal degradation, independent of GSK3β-mediated phosphorylation