Beta catenin is regulated by its subcellular distribution and mutant huntingtin status in Huntington's disease cell STHdhQ111/HdhQ111.

Dysregulation of gene expression at RNA and protein level is a hallmark of Huntington's disease (HD). Altered levels of microRNAs and beta catenin in HD were studied earlier; however, any direct involvement of full length, basally-expressing mutant huntingtin (Htt) remained to be elusive. Here we reported that the gain-of-function mutation of full-length basally-expressing Htt in HD cell Q111 (STHdhQ111/HdhQ111) upregulated microRNA-214 and decreased beta catenin & its transcriptional activity in an aggregate-independent manner. The result was quite opposite of the function of aggregate-forming mutant Htt fragment 83Q-DsRed. Here, we also reported an elevated level of beta catenin phosphorylation in Q111 cell compared to Q7 cell (SThdhQ7/HdhQ7). We showed that in Q111 cell (compared to Q7), beta catenin was more localized in the cytosol than that of the plasma membrane. This is significant as Gsk3beta phosphorylates beta catenin in the cytosol. Hence, for the first time, our study identified beta catenin localization and mutant Htt status as two key factors of beta catenin regulation in HD.

Inhibition of cancer progression by a novel trans-stilbene derivative through disruption of microtubule dynamics, driving G2/M arrest, and p53-dependent apoptosis.

Resveratrol, a trans-stilbene polyphenolic compound and its synthetic analogs are widely used bioactive molecules due to their remarkable chemo-preventive potential. Here, we have identified a novel synthetic trans-stilbene compound, Z-DAN-11 ((Z)-3-(3, 4-dimethoxyphenyl)-2-(3, 4, 5-trimethoxyphenyl) acrylonitrile) which shows remarkable efficacy in blocking tumor growth and progression both in vitro and in vivo. Z-DAN-11 inhibits proliferation of cancer cells in vitro through microtubule depolymerization that induced G2/M arrest and consequently leads to apoptotic cell death. More importantly, Z-DAN-11 shows limited cytotoxicity to normal cells as compared to cancer cells. Quite interestingly, we have found that Z-DAN-11-mediated ROS production helps in dramatic alteration in the mitochondrial redox status which critically contributes to the apoptosis. Mechanistic studies reveal that Z-DAN-11 induces the expression of pro-apoptotic proteins and decreases anti-apoptotic protein expression that decisively helps in the activation of caspase 8, caspase 9, and caspase 3, leading to cleavage of PARP1 and cell death via intrinsic and extrinsic pathways of apoptosis. Moreover, Z-DAN-11-mediated apoptosis of cancer cells is through a partial p53-dependent pathway, since both HCT116 p53-/- cells as well as p53-silenced cells (siRNA) were able to block apoptosis partially but significantly. Importantly, Z-DAN-11 also imparts its anti-tumorigenic effect by inhibiting clonogenic property and anchorage-independent growth potential of cancer cells at concentrations at least 10 times lower than that required for inducing apoptosis. Finally, in vivo study with immuno-competent syngeneic mice shows Z-DAN-11 to be able to impede tumor progression without any adverse side-effects. Hence, we identified a novel, synthetic trans-stilbene derivative with anti-tumorigenic potential which might tremendously help in devising potential therapeutic strategy against cancer.

Biomonitoring role of some cellular markers during heat stress-induced changes in highly representative fresh water mollusc, Bellamya bengalensis: Implication in climate change and biological adaptation.

Owing to increasing concern of global climate-change, temperature rise is of great interest which can be primarily evaluated from the seasonal variations in some organisms. Aquatic environment can be extremely stressful to its inhabitants because most of them are poikilothermous. In the present study, attempt was made to evaluate the biological effects of oxidative-stress and adaptive/antioxidant capacities during temperature variations (36-40 °C for 24hrs to 72hrs) in Bellamya bengalensis both in environmental and laboratory conditions by testing some biomarkers like HSP70, catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH) and glutathione reductase (GR). The biomarker potency of the molecules and the anti-oxidative metabolic-network was postulated and extrapolated to find its resemblance to the climate-change associated organismal variations. In a natural and eco-restored environment in the Eastern part of India, 10-20 fold increases in CAT, SOD and HSP70 protein expressions (Western blot results) were noticed in Bellamya paralleling to their increased enzymatic activities (gel zymogram studies) due to the seasonal (summer versus winter) temperature variation. It is evident from the consecutive three years' study that this variation resulted in the unfavorable physico-chemical changes of water quality parameters like dissolved oxygen, biochemical oxygen demand, alkalinity and consequently decreased the animal density in summer. And that was revived due to their higher reproduction-rate in post rainy/winter season when temperature normalizes resulting in a restoration of favorable environment. In laboratory condition, the reduced GR and increased GPx indicated the oxidative damage as evident by higher tissue MDA level following to higher mortality. Changes in SOD and CAT activities suggest activation of physiological mechanism to scavenge the ROS produced during heat stress. However, when mortality increased at different time points (36 °C - 72 h and 38 °C - 72 h), these enzyme activities also decreased as they failed to save the tissues from ROS. The results suggest that temperature variation does alter the active oxygen metabolism by modulating antioxidant enzyme activities, which can be used as biomarker to detect sub-lethal effects of climate change-associated pollution. The parity in environmental and laboratory experimental results may justify this laboratory experiment as model heat-stress experiment and indicate temperature as a universal stressor which alone or in combination with other water parameters initiates a consistent adapting behavior. The Bellamya bengalensis being the highest faunal representative in its habitat may serve as a good bioindicator species.

Benzo(a)pyrene Induced p53 Mediated Male Germ Cell Apoptosis: Synergistic Protective Effects of Curcumin and Resveratrol.

Benzo(a)pyrene (B(a)P) 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 reactive oxygen species (ROS) 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.

Human Prostate Cancer Hallmarks Map.

Human prostate cancer is a complex heterogeneous disease that mainly affects elder male population of the western world with a high rate of mortality. Acquisitions of diverse sets of hallmark capabilities along with an aberrant functioning of androgen receptor signaling are the central driving forces behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. These hallmark capabilities arise due to an intense orchestration of several crucial factors, including deregulation of vital cell physiological processes, inactivation of tumor suppressive activity and disruption of prostate gland specific cellular homeostasis. The molecular complexity and redundancy of oncoproteins signaling in prostate cancer demands for concurrent inhibition of multiple hallmark associated pathways. By an extensive manual curation of the published biomedical literature, we have developed Human Prostate Cancer Hallmarks Map (HPCHM), an onco-functional atlas of human prostate cancer associated signaling and events. It explores molecular architecture of prostate cancer signaling at various levels, namely key protein components, molecular connectivity map, oncogenic signaling pathway map, pathway based functional connectivity map etc. Here, we briefly represent the systems level understanding of the molecular mechanisms associated with prostate tumorigenesis by considering each and individual molecular and cell biological events of this disease process.

Resveratrol ameliorates benzo(a)pyrene-induced testicular dysfunction and apoptosis: involvement of p38 MAPK/ATF2/iNOS signaling.

Benzo(a)pyrene [B(a)P] is an environmental toxicant that alters the steroidogenic profile of testis and induces testicular dysfunction. In the present study, we have investigated the molecular signaling of B(a)P and the ameliorative potential of the natural aryl hydrocarbon receptor (AhR) antagonist and antioxidant, resveratrol, on B(a)P-induced male reproductive toxicity. Studies showed that B(a)P treatment resulted in p38 MAPK activation and increased inducible nitric oxide synthase (iNOS) production along with testicular apoptosis and steroidogenic dysfunction. Resveratrol cotreatment maintained testicular redox potential, increased serum testosterone level and enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3ßHSD, 17ßHSD) and prevented subsequent onset of apoptosis. Resveratrol cotreatment resulted inhibition of testicular cytochrome P4501A1 (CYP1A1) expression, which is the major B(a)P metabolizing agent for BPDE-DNA adduct formation. Resveratrol also significantly decreased the B(a)P-induced AhR protein level, its nuclear translocation and subsequent promoter activation, thereby decreased the expression of CYP1A1. Resveratrol also down-regulated B(a)P-induced testicular iNOS production through suppressing the activation of p38 MAPK and ATF2, thus improved the oxidative status of the testis and prevented apoptosis. Our findings cumulatively suggest that resveratrol inhibits conversion of B(a)P into BPDE by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

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.

Piroxicam, a traditional non-steroidal anti-inflammatory drug (NSAID) causes apoptosis by ROS mediated Akt activation.

BACKGROUND: Piroxicam (Px) belongs to the oxicam group of the non-steroidal anti-inflammatory drugs (NSAIDs) and have been shown to exert chemopreventive and chemotherapeutic effects in animal models and cultured animal cells. However, little is known about the mode of action of Px and its cellular targets. METHODS: We explored the role of Px, in triggering apoptosis and examined the involvement of upstream cellular mechanisms in apoptosis induction by Px. RESULTS AND DISCUSSION: Our studies with human breast cancer cells MCF-7 show that Px induces reactive oxygen species (ROS) generation along with apoptotic cell death. ROS release lead to Akt activation. On evaluation it became evident that ROS mediated apoptosis induction was due to Akt activation (hyper phosphorylation). Silencing the expression of Akt using siRNA and a specific Akt inhibitor, triciribine further confirmed the findings. However Px failed to cause ROS generation, cell death or Akt phosphorylation in another human breast cancer cells MDA-MB-231 which is estrogen receptor negative and more aggressive compared to MCF-7 cells. This suggests that Px has cell type specific effects. Thus we revealed for the first time that Px can induce apoptosis by ROS mediated Akt hyperphosphorylation/activation.

Beta catenin is degraded by both caspase-3 and proteasomal activity during resveratrol-induced apoptosis in HeLa cells in a GSK3ß-independent manner.

Increased activity of ß-catenin, an important transcriptional activator for survival and proliferation-associated genes has been linked with many cancers. We examined whether ß-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 ß-catenin levels remained unchanged for 48 h during resveratrol exposure. However, extranuclear cell lysate ß-catenin underwent a decrease at a late stage of apoptosis namely at 36-48 h. Alterations in the phosphorylation status of Akt/GSK3ß were not observed during resveratrol-induced apoptosis. Furthermore, inhibition of GSK3ß activity which is. largely responsible for ß-catenin degradation failed to influence ß-catenin stability. However, inhibition of caspase-3 activity prevented the decline in ß-catenin levels at 36-48 h of resveratrol exposure. Lactacystin, a proteosomal inhibitor also prevented the degradation of ß-catenin by resveratrol. In conclusion, resveratrol induced apoptosis in HeLa cells in an Akt/GSK3ß-independent manner and down-regulated ß-catenin levels during apoptosis through action of caspase-3 and proteasomal degradation, independent of GSK3ß-mediated phosphorylation.

Micro RNA-214 contributes to proteasome independent downregulation of beta catenin in Huntington's disease knock-in striatal cell model STHdhQ111/Q111.

Role of beta catenin in Huntington's disease (HD) is not clear. Previous studies on HD reported varied levels of beta catenin. In the present study we showed that beta catenin is post transcriptionally down-regulated in mutant huntingtin knock-in cell model STHdhQ111/Q111. This in turn leads to decreased level of wnt/beta catenin responsive genes. We observed that Gsk3beta or Gsk3beta (phospho Ser 9) is unaltered in HD and this down-regulation of beta catenin is independent of proteasomal degradation. Finally, we showed that the overexpression of miR-214 leads to the down-regulation of beta catenin at protein level only and reduces its transcriptional activity. We concluded that, miR-214 contributes to the processes that result in proteasome independent post transcriptional down-regulation of beta catenin in STHdhQ111/Q111, probably through inhibition of protein synthesis from beta catenin mRNA.

Molecular and functional characterisation of a stress responsive cysteine protease, EhCP6 from Entamoeba histolytica.

Entamoeba histolytica cysteine protease 6 (EhCP6) is a stress responsive cysteine protease that is upregulated in response to heat shock and during pathogen invasion of the host tissue. In the present study an attempt has been made to express and purify recombinant EhCP6 in order to gain insights into its biochemical properties. The recombinant and refolded protein has been shown to undergo autoproteolysis in the presence of DTT and SDS to give rise to ∼25kDa mature form. The mature form of the protein was found to exhibit a protease activity that is sensitive to E-64, a specific cysteine protease inhibitor. In silico homology modelling of EhCP6 revealed that the protein exhibits conservation of almost all the major structural features of cathepsin-L like cysteine proteases. Further in vivo studies are needed to decipher the function of the protein in response to different stressed conditions.

Assessment of thermal stress adaptation by monitoring Hsp70 and MnSOD in the freshwater gastropod, Bellamya bengalensis (Lamark 1882).

Expression of the stress biomarkers 70-kDa heat shock proteins (Hsp70) and manganese superoxide dismutase (MnSOD) was measured as the molecular basis of adaptive response against increased experimental temperatures (32-40 °C for a span of 24-72 h) on the fresh water molluscan species, Bellamya bengalensis (Lamark 1882). The experimental snail specimens were collected during summer and winter seasons from two contrasting wetlands: an ecorestored (free from human interference) site (SI) and other experiencing anthropogenic stresses (SII). The mortality rate of the B. bengalensis and the immunoblotting of MnSOD and Hsp70 of their digestive glands were performed at regular intervals during the period of heat stress. The SI provided a lower stress environment based on physicochemical parameters such as pH, dissolved oxygen (DO), biological oxygen demand (BOD), chemical oxygen demand (COD), and alkalinity for the survival of test species, although both sites experienced mortality due to thermal stresses. The parity in protein expressions displayed a uniform mode of adaptive impact to temperature elevations in both field and laboratory exposure. The Hsp70 expression was minimal at lower thermal stress, but increased with a rise in temperature. It is very likely that higher Hsp70 levels are not directly related to survival or adaptation. In contrast, MnSOD levels appeared to be an indicator of adaptive responses vis-a-vis survival of the animals. So, the expression levels of a universal free radical scavenger like MnSOD are recognized as a potential biomarker in a bioindicator species like Bellamya.

Synthesis and evaluation of triazole linked glycosylated 18ß-glycyrrhetinic acid derivatives as anticancer agents.

A series of glycosyl triazol linked 18ß-glycyrrhetinic acid (GA) derivatives have been synthesized using 1,3-dipolar cycloaddition reaction of per-O-acetylated glycosyl azide derivatives (4a-h) with propargyl ester of 18ß-glycyrrhetinic acid (GA) (2 and 3) following the concept of 'Click chemistry'. The synthesized triazole derivatives were de-O-acetylated to furnish compounds (7a-h and 8a-c) with free hydroxyl groups in the carbohydrate moieties, which were evaluated for their anticancer potential against human cervical cancer cells (HeLa) and normal kidney epithelial (NKE) cells. GA (1), compound 7d, compound 7g and compound 8c showed promising anticancer activities.

Drought resistance in rice seedlings conferred by seed priming : role of the anti-oxidant defense mechanisms.

Seed priming is a method by which seeds are subjected to different stress conditions to impart stress adaptation in seedlings germinating and growing under stressful situations. Drought stress is a major reason behind failure of crops. We studied the effects of hydropriming, dehydration priming (induced by PEG), and osmopriming (induced by NaCl and KH(2)PO(4)) on subsequent germination, growth and anti-oxidant defense mechanisms of 2-week-old rice seedlings under continuing dehydration stress. Unprimed seeds grown in PEG showed significantly lower germination and growth along with significantly higher reactive oxygen species (ROS) and lipid peroxidation levels. Among the priming methods, 5 % PEG priming was found to be the best in terms of germination and growth rate along with the lowest amount of ROS and lipid peroxidation (malondialdehyde [MDA]) values. MDA levels were reduced significantly by all of the priming methods. Hence, reduction of lipid peroxidation may be a key factor underlying the drought tolerance produced by the priming treatments. Glutathione peroxidase (GPX) activity seemed to bear an excellent correlation with oxidative stress resistance through seed priming. The PEG priming produced minimum peroxidative damage and superior germination and growth rate along with efficient GPX activity, overexpressed MnSOD and maintenance of HSP70 expression in normal as well as in drought condition. Therefore, in PEG-primed seeds the existence of robust protective mechanisms is definitely indicated.

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 H(2)O(2) treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H(2)O(2) treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H(2)O(2) treatment reduced parasite viability significantly but heat shock and 0.5 mM H(2)O(2) 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.

Hydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolytica.

The microaerophilic intestinal parasitic protozoan Entamoeba histolytica has been previously shown to be highly susceptible to oxidative stress induced by hydrogen peroxide. However the mechanism of cell death was not investigated. Studies presented in this paper demonstrate several morphological features in the parasite when exposed to H(2)O(2) which are identical to metazoan apoptotic phenotype indicating a possible apoptosis-like cell death exhibited by E. histolytica in response to H(2)O(2) treatment. Trophozoite cell shrinkage, DNA fragmentation, phosphatidyl serine externalization and increased endogenous reactive oxygen species level have been observed in the protozoan parasite when exposed to 2.0mM H(2)O(2) for different time periods. Although the parasite genome is completely devoid of any of the homologues of mammalian caspases it still codes for a huge number of cysteine proteases which may take over the apoptotic function of the caspases. But the present study indicates the existence of a cysteine protease independent programmed cell death in the parasite since E-64 the specific cysteine protease inhibitor could not rescue the cells from H(2)O(2) induced apoptosis-like cell death.

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.

BACKGROUND AND AIMS: Plants can withstand many abiotic stresses. Stress adaptation through retention of imprints of previous stress exposure has also been described in plants. We have characterized the imprint or memory of adaptive stress responses of rice seedlings to arsenic (As) and heat stress. METHODOLOGY: Two-week-old rice seedlings (both with and without As) were given a 45 °C heat shock for 3 h. While under heat shock, the leafy portion of the seedlings was harvested at regular intervals. Subsequently, the seedlings were kept at room temperature for recovery and sampling continued over 3 h. Total RNA and protein were extracted from the leafy portion of the seedlings and complementary DNA (cDNA) was prepared from total RNA. The cDNA was used as a template for the polymerase chain reaction to identify the transcription level of HSP70. Protein extracted from the seedlings was western-blotted. HSP70 and actin (loading control) antibodies were used to recognize the proteins on the same blot. PRINCIPAL RESULTS: Our studies reveal that HSP70, a cellular chaperone gene, is over-expressed at the mRNA and protein levels when rice seedlings are exposed to As and heat. The effect is cumulative and increases with the duration of stress for 3 h. During 3 h recovery from heat stress at ambient temperatures for 3 h, the chaperone remains expressed at higher levels in plants pre-exposed to As. CONCLUSIONS: Our findings demonstrate a retention of the imprint of previous stress exposure, perhaps through sustained activation of the signalling pathways upstream of over-expression of HSP70. Furthermore, stress-induced HSP70 expression was additive/cumulative for continued exposure to similar or different kinds of stress, indicating that a commonality of signal transduction networks is adopted when plants experience more than one stress.

Heat stress upregulates chaperone heat shock protein 70 and antioxidant manganese superoxide dismutase through reactive oxygen species (ROS), p38MAPK, and Akt.

Chinese hamster lung fibroblasts V79 cells were treated with heat stress for 4 weeks with short duration (15 min) heat shock every alternate day in culture. It was observed that Hsp 70 and the antioxidant enzyme MnSOD became overexpressed during the chronic heat stress period. Both p38 MAPK and Akt became phosphorylated by chronic heat stress exposure. Simultaneous exposure to SB203580, a potent and specific p38MAPK inhibitor drastically inhibited the phosphorylation of p38MAPK and Akt. Furthermore, exposure to SB203580 also blocked the increase in Hsp70 and MnSOD levels and the elevated SOD activity brought about by chronic heat stress. Heat shock factor 1 (HSF1) transcriptional activity and nuclear translocation of HSF1 were prominently augmented by chronic heat stress, and this amplification is markedly reduced by concomitant exposure to SB203580. Also, activations of p38MAPK and Akt and upregulations of Hsp70 and MnSOD were observed on exposure to heat shock for a single exposure of longer duration (40 min). siRNA against p38MAPK notably reduced Akt phosphorylation by single exposure to heat stress and drastically diminished the rise in Hsp70 and MnSOD levels. Similarly, siRNA against Akt also eliminated the augmentation in Hsp70 and MnSOD levels but p38MAPK levels remained unaffected. Heat stress produced reactive oxygen species (ROS) in V79 fibroblasts. N-acetyl cysteine blocked the increase in phosphorylation of p38MAPK, amplification of Hsp70, and MnSOD levels by heat stress. Therefore, we conclude that heat stress-activated p38MAPK which in turn activated Akt. Akt acted downstream of p38MAPK to increase Hsp70 and MnSOD levels.Concise summary: Thermal injury of the skin over a long period of time has been associated with development of cancerous lesions. Also, in many cancers, the cytoprotective genes Hsp70 and MnSOD have been found to be overexpressed. Therefore, we considered it important to identify the signaling elements upstream of the upregulated survival genes in heat stress. We conclude that heat stress activated p38MAPK which in turn activated Akt. Akt mediated an augmentation in Hsp70 and MnSOD levels working downstream of p38MAPK.

Pro-survival effects of repetitive low-grade oxidative stress are inhibited by simultaneous exposure to Resveratrol.

V79 lung fibroblasts were subjected to repetitive oxidative stress in culture through exposures to 30 microM H(2)O(2) for 4 weeks. Repetitively stressed cells were found to be significantly resistant to apoptosis-inducing agent such as ultraviolet radiation (UVR). Concurrent treatment with Resveratrol completely restored the normal apoptotic response after UVR. p38MAPK became dually phosphorylated during the stress period. Akt also became phosphorylated on Ser(473) in cells subjected to repetitive oxidative stress. In these cells, NFkappaB p65 became phosphorylated and appreciable nuclear localization of p65 was observed. NFkappaB transcriptional activity also became augmented during repetitive stress. Treatment of the repetitively stressed cells concurrently with Resveratrol or SB203580, a p38MAPK inhibitor, robustly blocked activation of p38MAPK, NFkappaB transcriptional activity, phosphorylation and nuclear localization of p65, and Akt phosphorylation. Pre-exposure to short interfering RNA (si RNA) to p38MAPK, resulted in a blockage of the Akt and NFkappaB p65 phosphorylation. However, inhibition of Akt activity through PI3 kinase inhibitor LY294002 did not result in obstruction of p38MAPK phosphorylation by H(2)O(2). Also, Resveratrol was effective as an antioxidant in counteracting a rise in reactive oxygen species (ROS) and p38MAPK activation by H(2)O(2) was completely blocked by antioxidant N-acetyl cysteine (NAC). We conclude that Resveratrol acts as an antioxidant and completely reverses the anti-apoptotic effects of repetitive stress by blocking oxidative stress-induced p38MAPK activation which is the key regulatory step for the activation of down-stream survival elements Akt and NFkappaB.