Heat stress triggers apoptosis by impairing NF-kappaB survival signaling in malignant B cells.

Nuclear factor-kappaB (NF-kappaB) is involved in multiple aspects of oncogenesis and controls cancer cell survival by promoting anti-apoptotic gene expression. The constitutive activation of NF-kappaB in several types of cancers, including hematological malignancies, has been implicated in the resistance to chemo- and radiation therapy. We have previously reported that cytokine- or virus-induced NF-kappaB activation is inhibited by chemical and physical inducers of the heat shock response (HSR). In this study we show that heat stress inhibits constitutive NF-kappaB DNA-binding activity in different types of B-cell malignancies, including multiple myeloma, activated B-cell-like (ABC) type of diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma presenting aberrant NF-kappaB regulation. Heat-induced NF-kappaB inhibition leads to rapid downregulation of the anti-apoptotic protein cellular inhibitor-of-apoptosis protein 2 (cIAP-2), followed by activation of caspase-3 and cleavage of the caspase-3 substrate poly(adenosine diphosphate ribose)polymerase (PARP), causing massive apoptosis under conditions that do not affect viability in cells not presenting NF-kappaB aberrations. NF-kappaB inhibition by the proteasome inhibitor bortezomib and by short-hairpin RNA (shRNA) interference results in increased sensitivity of HS-Sultan B-cell lymphoma to hyperthermic stress. Altogether, the results indicate that aggressive B-cell malignancies presenting constitutive NF-kappaB activity are sensitive to heat-induced apoptosis, and suggest that aberrant NF-kappaB regulation may be a marker of heat stress sensitivity in cancer cells.

Activation of I kappa b kinase by herpes simplex virus type 1. A novel target for anti-herpetic therapy.

Herpes simplex viruses (HSV) are ubiquitous pathogens causing a variety of diseases ranging from mild illness to severe life-threatening infections. HSV utilize cellular signaling pathways and transcription factors to promote their replication. Here we report that HSV type 1 (HSV-1) induces persistent activation of transcription factor NF-kappa B, a critical regulator of genes involved in inflammation, by activating the I kappa B kinase (IKK) in the early phase of infection. Activated NF-kappa B enhances HSV-1 gene expression. HSV-1-induced NF-kappa B activation is dependent on viral early protein synthesis and is not blocked by the anti-herpetic drug acyclovir. IKK inhibition by the anti-inflammatory cyclopentenone prostaglandin A(1) blocks HSV-1 gene expression and reduces virus yield by more than 3000-fold. The results identify IKK as a potential target for anti-herpetic drugs and suggest that cyclopentenone prostaglandins or their derivatives could be used in the treatment of HSV infection.

Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase.

NF-kappaB is a critical activator of genes involved in inflammation and immunity. Pro-inflammatory cytokines activate the IkappaB kinase (IKK) complex that phosphorylates the NF-kappaB inhibitors, triggering their conjugation with ubiquitin and subsequent degradation. Freed NF-kappaB dimers translocate to the nucleus and induce target genes, including the one for cyclo-oxygenase 2 (COX2), which catalyses the synthesis of pro-inflammatory prostaglandins, in particular PGE. At late stages of inflammatory episodes, however, COX2 directs the synthesis of anti-inflammatory cyclopentenone prostaglandins, suggesting a role for these molecules in the resolution of inflammation. Cyclopentenone prostaglandins have been suggested to exert anti-inflammatory activity through the activation of peroxisome proliferator-activated receptor-gamma. Here we demonstrate a novel mechanism of antiinflammatory activity which is based on the direct inhibition and modification of the IKKbeta subunit of IKK. As IKKbeta is responsible for the activation of NF-kappaB by pro-inflammatory stimuli, our findings explain how cyclopentenone prostaglandins function and can be used to improve the utility of COX2 inhibitors.

Delta(12)-prostaglandin J(2) is a potent inhibitor of influenza A virus replication.

9-Deoxy-Delta(9),Delta(12)-13,14-dihydro-prostaglandin D(2) (Delta(12)-PGJ(2)), a natural cyclopentenone metabolite of prostaglandin D(2), is shown to possess therapeutic efficacy against influenza A virus A/PR8/34 (H1N1) infection in vitro and in vivo. The results indicate that the antiviral activity is associated with induction of cytoprotective heat shock proteins and suggest novel strategies for treatment of influenza virus infection.

Heat shock factors and the control of the stress response.

Living cells are continually challenged by conditions which cause acute and chronic stress. To adapt to environmental changes and survive different types of injuries, eukaryotic cells have evolved networks of different responses which detect and control diverse forms of stress. One of these responses, known as the heat shock response, has attracted a great deal of attention as a universal fundamental mechanism necessary for cell survival under a variety of unfavorable conditions. In mammalian cells, the induction of the heat shock response requires the activation and translocation to the nucleus of one or more heat shock transcription factors which control the expression of a specific set of genes encoding cytoprotective heat shock proteins. The discovery that the heat shock response is turned on under several pathological conditions and contributes to establish a cytoprotective state in a variety of human diseases, including ischemia, inflammation, and infection, has opened new perspectives in medicine and pharmacology, as molecules activating this defense mechanism appear as possible candidates for novel cytoprotective drugs. This article focuses on the regulation and function of the heat shock response in mammalian cells and discusses the molecular mechanisms involved in its activation by stress and bioactive cyclopentenone prostanoids, as well as its interaction with nuclear factor kappaB, a stress-regulated transcription factor with a pivotal role in inflammation and immunity.

Induction of ferritin and heat shock proteins by prostaglandin A1 in human monocytes. Evidence for transcriptional and post-transcriptional regulation.

Prostaglandins of the A type (PGA) exert a cytoprotective activity during hyperthermia and virus infection. This effect is associated with induction of heat shock proteins (HSP) in mammalian cells. We now report that, in human monocytes, PGA1 is able to induce the synthesis of the iron-binding, redox-regulated protein ferritin. L-chain ferritin induction is consequent to a substantial increase in the accumulation of L-chain ferritin transcripts in PGA1-treated cells, whereas H-chain ferritin is regulated post-transcriptionally, consequently to reduction of iron-regulatory protein binding to iron-responsive elements in ferritin mRNA. Ferritin induction is specific for cyclopentenone prostaglandins (PGA1, PGA2, PGJ2, Delta12-PGJ2), whereas other arachidonic acid (AA) metabolites have no effect. In human monocytes, PGA1 also induces heat shock gene transcription via heat shock factor activation, as well as the synthesis of the oxidative-stress protein heme oxygenase (HOS). Differently from HSP, the induction of ferritin by PGA1 is specific for monocytes. Monocytes/macrophages play a pivotal role in inflammation, controlling iron metabolism and releasing a variety of mediators, including proinflammatory reactive oxygen species (ROS), cytokines and AA metabolites. As ferritin, together with hsp70 and HO, plays a key role in protection from oxidant damage, these results suggest that PGA1 may have cytoprotective activity also during oxidative injury.

Antiviral effect of hyperthermic treatment in rhinovirus infection.

Human rhinoviruses (HRV) are recognized as the major etiologic agents for the common cold. Starting from the observation that local hyperthermic treatment is beneficial in patients with natural and experimental common colds, we have studied the effect of brief hyperthermic treatment (HT) on HRV replication in HeLa cells. We report that a 20-min HT at 45 degrees C is effective in suppressing HRV multiplication by more than 90% when applied at specific stages of the virus replication cycle. Synthesis of virus proteins is not affected by HT, indicating that the target for treatment is a posttranslational event. The antiviral effect is a transient cell-mediated event and is associated with the synthesis of the 70-kDa heat shock protein hsp70. Unlike poliovirus, rhinovirus infection does not inhibit the expression of hsp70 induced by heat. The possibility that hsp70 could play a role in the control of rhinovirus replication is suggested by the fact that a different class of HSP inducers, the cyclopentenone prostaglandins PGA1 and delta 12-PGJ2, were also effective in inhibiting HRV replication in HeLa cells. Inhibition of hsp70 expression by actinomycin D prevented the antiviral activity of prostaglandins in HRV-infected cells. These results indicate that the beneficial effect of respiratory hyperthermia may be mediated by the induction of a cytoprotective heat shock response in rhinovirus-infected cells.

Induction of the heat-shock response by antiviral prostaglandins in human cells infected with human immunodeficiency virus type 1.

Cyclopentenone prostaglandins inhibit the replication of several DNA and RNA viruses, including retroviruses. The antiviral activity has been associated with the induction of a 70-kDa heat-shock protein (HSP70), via activation of the heat-shock transcription factor (HSF) in infected cells. In the present study we investigated the effect of prostaglandin A1 (PGA1) on the regulation of HSP70 gene expression as well as on viral RNA and protein synthesis in CEM-SS cells during acute infection with human immunodeficiency virus type 1 (HIV-1). We report that HIV-1 infection does not alter HSF activation by PGA1, whereas it causes an increase in intracellular HSP70 mRNA levels, as a result of enhanced HSP70 mRNA stability. We also show that, as reported in studies of different virus/host cell models, PGA1 inhibits HIV-1 replication by acting at multiple levels during HIV-1 infection. In addition to the previously reported block of HIV-1 mRNA transcription, PGA1 was also found to inhibit viral protein synthesis. These results, together with the fact that prostaglandins are used clinically in the treatment of several diseases, open new perspectives in the search for novel antiretroviral drugs.

Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection.

Molecular chaperones protect proteins against environmental and physiologic stress and from the deleterious consequences of an imbalance in protein homeostasis. Many of these stresses, if prolonged, result in defective development and pathologies associated with a diverse array of diseases due to tissue injury and repair including stroke, myocardial reperfusion damage, ischemia, cancer, amyloidosis, and other neurodegenerative diseases. We discuss the molecular nature of the stress signals, the mechanisms that underlie activation of the heat shock response, the role of heat shock proteins as cytoprotective molecules, and strategies for pharmacologically active molecules as regulators of the heat shock response.

Inhibition of rotavirus replication by prostaglandin A: evidence for a block of virus maturation.

Rotaviruses are recognized as the leading cause of severe viral gastroenteritis in young children and in immunocompromised patients. Cyclopentenone prostaglandins possess antiviral activity against several single-strand RNA viruses; therefore, the effect of prostaglandin A1 (PGA1) on SA-11 simian rotavirus infection was investigated in cultured cells. PGA1 potently inhibited SA-11 rotavirus replication. Whereas it did not affect virus adsorption or penetration, PGA1 partially inhibited VP4 and VP7 synthesis and selectively reduced glucosamine incorporation into the NSP4 viral enterotoxin. Electron microscopy analysis showed that, despite normal formation of cytoplasmic inclusions and budding of particles into the rough endoplasmic reticulum, virus maturation was impaired in PGA1-treated cells, with most of the virus particles remaining in the membrane-enveloped intermediate form. Because prostaglandins are used clinically as cytoprotective drugs for gastric ulcers, these observations offer new perspectives in the search for therapeutic agents for rotavirus-induced gastroenteritis.

Activation of the heat shock factor 1 by serine protease inhibitors. An effect associated with nuclear factor-kappaB inhibition.

Heat shock proteins (HSPs) have a cytoprotective role in several human diseases, including ischemia and viral infection. Nuclear factor-kappaB (NF-kappaB) is a critical regulator of inflammation and virus replication. Here we report that a class of serine protease inhibitors with NF-kappaB-inhibitory activity are potent HSP inducers via activation of heat shock transcription factor 1 (HSF1) in human cells. 3,4-Dichloroisocoumarin, the most effective compound, rapidly induces HSF1 DNA binding activity and phosphorylation, leading to transcription and translation of heat shock genes for a period of several hours. HSF1 activation is independent of de novo protein synthesis and is correlated in a concentration- and time-dependent manner with NF-kappaB inhibition. Cysteine protease inhibitors E64 and calpain inhibitor II, which do not block NF-kappaB activation, do not induce HSF DNA binding activity. HSP induction by 3,4-dichloroisocoumarin is associated with antiviral activity during rhabdovirus infection. These results identify a new class of HSP inducers and indicate a link between the regulatory pathways of HSF and NF-kappaB, suggesting novel strategies to simultaneously switch on cytoprotective genes and down-regulate inflammatory and viral genes.

Antiviral activity of cyclopentenone prostanoids.

Cyclopentenone prostanoids inhibit virus replication by turning on an intracellular defence response that involves the induction of cytoprotective heat-shock proteins, the modification of viral glycoprotein maturation and the control of NF-kappa B activation. These molecules represent an interesting model for the development of novel antiviral drugs that can affect different targets during the virus life cycle.

Inhibition of nuclear factor kappa B by prostaglandin A1: an effect associated with heat shock transcription factor activation.

Prostaglandins (PGs) function as intracellular signal mediators in the regulation of a variety of physiological and pathological processes, including inflammation and immune responses. Cyclopentenone PGs are characterized by antiviral activity against several viruses, including human immunodeficiency virus type 1 (HIV-1), and by the ability to induce heat shock protein expression through activation of the heat shock transcription factor. Here we report that PGA1 is a potent inhibitor of nuclear factor-kappa B (NF-kappa B) activation in human cells and of NF-kappa B-dependent HIV-1 transcription in long terminal repeat-chloramphenicol acetyl-transferase transient transfection experiments. PGA1 acts by inhibiting phosphorylation and preventing degradation of the NF-kappa B inhibitor I kappa B-alpha. Inhibition does not require protein synthesis, is dependent on the presence of a reactive cyclopentenonic moiety, and is associated with heat shock transcription factor activation. Because NF-kappa B is critically involved in the activation of immunoregulatory and viral genes, inhibition of its activity could be a major component of the immunosuppressive and antiviral activity of PGs.

2-Cyclopenten-1-one, a new inducer of heat shock protein 70 with antiviral activity.

The cytoprotective role of heat shock proteins (HSP) described in variety of human diseases, including ischemia, inflammation, and infection, suggests new therapeutic strategies relying upon the development of drugs that selectively turn on heat shock genes. Cyclopentenone prostaglandins, which contain an alpha, beta-unsaturated carbonyl group in the cyclopentane ring and possess antiviral activity against several RNA and DNA viruses, were shown to function as signal for HSP synthesis in a nonstressful situation in a variety of mammalian cells. We now report that 2-cyclopenten-1-one selectively induces the expression of the 70-kDa HSP (HSP70) in human cells, through cycloheximide-sensitive activation of heat shock transcription factor 1 (HSF1). The alpha, beta-unsaturated carbonyl group is the key structure triggering HSF1 activation. Induction is associated with antiviral activity during infection with vesicular stomatitis virus. These results identify the molecular structure of natural prostaglandins responsible for HSF1 activation and open new perspectives in the search for novel antiviral and cytoprotective drugs.

Inhibition of HSP70 expression by calcium ionophore A23187 in human cells. An effect independent of the acquisition of DNA-binding activity by the heat shock transcription factor.

Heat shock proteins (HSPs) are induced in mammalian cells in a variety of pathophysiological states and have an important role in cytoprotection in vitro and in vivo. In this study, we report that the calcium ionophore A23187, a glucose-regulated protein (GRP) inducer, dramatically inhibits HSP70 synthesis and HSP70 mRNA transcription after induction by heat shock, sodium arsenite, or prostaglandin A1 treatment in human K562 cells. A23187 does not suppress, and it actually prolongs, the DNA-binding activity of the human heat shock transcription factor (HSF), while it alters HSF1 phosphorylation in heat shock-treated cells. To inhibit HSP70 expression, A23187 needs to be present during heat shock, while treatment before or after heat shock does not affect HSP70 mRNA transcription. The GRP inducer thapsigargin, which specifically inhibits the endoplasmic reticulum Ca2+-ATPase, has no effect on heat-induced HSP70 synthesis, indicating that A23187 inhibitory activity is not due to depletion of intracellular calcium stores and is independent of the concomitant induction of GRP genes. Inhibition of HSP70 expression is correlated with alterations in HSF1 phosphorylation in heat-shocked cells, but not in sodium arsenite-treated cells, indicating that different mechanisms may be involved in mediating A23187 inhibitory activity.

Inhibition of HIV-1 replication by cyclopentenone prostaglandins in acutely infected human cells. Evidence for a transcriptional block.

Cyclopentenone prostaglandins (PGs) inhibit virus replication in several DNA and RNA virus models, in vitro and in vivo. In the present report we demonstrate that the cyclopentenone prostaglandins PGA(1) and PGJ(2) at nontoxic concentrations can dramatically suppress HIV-1 replication during acute infection in CEM-SS cells. PGs did not affect HIV-1 adsorption, penetration, reverse transcriptase activity nor viral DNA accumulation in HIV-1 infected cells. A dramatic reduction in HIV-1 mRNA levels was detected up to 48-72 h after infection (p.i.) in PG-treated cells, and HIV-1 protein synthesis was greatly reduced by a single PG-treatment up to 96 h p.i. Repeated PGA(1)-treatments were effective in protecting CEM-SS cells by the cytopathic effect of the virus, and in dramatically reducing HIV-1 RNA levels up to 7 d after infection. The antiviral effect was not mediated by alterations in the expression of alpha-, beta-, or gamma-interferon,TNFalpha, TNFbeta, IL6, and IL10 in HIV-infected CEM-SS cells. The fact that prostaglandins are used clinically in the treatment of several diseases, suggests a potential use of cyclopentenone PGs in the treatment of HIV-infection.

Effect of combined alpha IFN and prostaglandin A1 treatment on vesicular stomatitis virus replication and heat shock protein synthesis in epithelial cells.

The antiviral activity of prostaglandin A (PGA) and interferons (IFNs) has been widely described. In the present report, we investigated the effect of combined alpha IFN and PGA1 treatment on vesicular stomatitis virus (VSV) replication and on heat shock protein (HSP) induction in monkey epithelia cells. In uninfected cells, PGA1 caused a dose-dependent induction of HSP70, HSP90 and HSP110, while alpha IFN did not affect HSP synthesis. Alpha-IFN suppressed VSV replication dose-dependently, even when cells were treated after virus infection. VSV protein synthesis was not affected by alpha IFN, indicating a block at the level of virus assembly or maturation. PGA1 caused a dose-dependent inhibition of VSV replication, and suppressed VSV protein synthesis at concentrations which induced the synthesis of high levels of HSP70. The combined treatment with low doses of alpha IFN or PGA1, which only moderately inhibited VSV replication when administered separately, was found to suppress VSV production by more than 95%, and resulted in a 3-fold increase of HSP70 synthesis as compared to PGA1 alone. These results demonstrate a co-operative effect of PGA1 and alpha IFN against VSV infection and suggest that alpha IFN can potentiate the cellular response to HSP induction in virus-infected cells.

Inhibition of poliovirus replication by prostaglandins A and J in human cells.

Cyclopentenone prostaglandins (PGs) inhibit the replication of a wide variety of enveloped DNA and RNA viruses. The antiviral activity is associated with alterations in the synthesis, maturation, and intracellular translocation of viral proteins. In the present report, we describe the effects of cyclopentenone PGs PGA1 and delta 12-PGJ2 on poliovirus (PV) replication in HeLa cells. Both PGs were found to inhibit PV replication dose dependently. Virus yield was significantly reduced at nontoxic concentrations, which did not suppress RNA or protein synthesis in uninfected or PV-infected cells. Both the pattern of PV proteins synthesized and the kinetics of viral protein synthesis and degradation appeared to be similar in PGA1-treated cells and control cells. Antiviral PGs have been shown to selectively inhibit virus protein synthesis during the replication of several viruses, including vesicular stomatitis virus (VSV), and this effect has been recently associated with the induction of a 70-kDa heat shock protein (HSP70). PGA1 and delta 12-PGJ2 were found to induce HSP70 synthesis in uninfected or VSV-infected HeLa cells. PV infection was found to inhibit PG-induced HSP70 synthesis in these cells, suggesting that the lack of ability of cyclopentenone PGs to block PV protein synthesis could be related to an impaired heat shock response in PV-infected cells. The finding that PV protein synthesis was not inhibited by PGs suggests that cyclopentenone PGs could interfere with a late event in the virus replication cycle, such as protein assembly and maturation of PV virions.