M. Gabriella Santoro

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.

NF-κB and virus infection: Who controls whom

Among the different definitions of viruses, ‘pirates of the cell’ is one of the most picturesque, but also one of the most appropriate. Viruses have been known for a long time to utilize a variety of strategies to penetrate cells and, once inside, to take over the host nucleic acid and protein synthesis machinery to build up their own components and produce large amounts of viral progeny. As their genomes carry a minimal amount of information, encoding only a few structural and regulatory proteins, viruses are largely dependent on their hosts for survival; however, despite their apparent simplicity, viruses have evolved different replicative strategies that are regulated in a sophisticated manner. During the last years, the study of the elaborate relationship between viruses and their hosts has led to the understanding of how viral pathogens not only are able to alter the host metabolism via their signaling proteins, but are also able to hijack cellular signaling pathways and transcription factors, and control them to their own advantage. In particular, the nuclear factor‐κB (NF‐κB) pathway appears to be an attractive target for common human viral pathogens. This review summarizes what is known about the control of NF‐κB by viruses, and discusses the possible outcome of NF‐κB activation during viral infection, which may benefit either the host or the pathogen.

Thiazolides, a new class of anti-influenza molecules targeting viral hemagglutinin at the post-translational level

The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, represents a serious threat to global human health. Efforts to control emerging influenza strains focus on surveillance and early diagnosis, as well as development of effective vaccines and novel antiviral drugs. Herein we document the anti-influenza activity of the anti-infective drug nitazoxanide and its active circulating-metabolite tizoxanide and describe a class of second generation thiazolides effective against influenza A virus. Thiazolides inhibit the replication of H1N1 and different other strains of influenza A virus by a novel mechanism: they act at post-translational level by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion, thus impairing hemagglutinin intracellular trafficking and insertion into the host plasma membrane, a key step for correct assembly and exit of the virus from the host cell. Targeting the maturation of the viral glycoprotein offers the opportunity to disrupt the production of infectious viral particles attacking the pathogen at a level different from the currently available anti-influenza drugs. The results indicate that thiazolides may represent a new class of antiviral drugs effective against influenza A infection.

Effect of nitazoxanide for treatment of severe rotavirus diarrhoea: randomised double-blind placebo-controlled trial

BACKGROUND Rotavirus is a leading cause of morbidity and mortality in children younger than 5 years, but there is no effective treatment. We assessed the activity of nitazoxanide, a broad-spectrum anti-infective drug, against rotavirus in cell culture and in a clinical trial in paediatric patients hospitalised with severe rotavirus diarrhoea. METHODS We did a randomised double-blind placebo-controlled trial in 50 children admitted to the Cairo University Childrens Hospital between June 15 and Aug 23, 2005, with severe rotavirus diarrhoea. 38 children aged 5 months to 7 years (median age 11 months) with rotavirus as the sole identified cause of gastroenteritis were enrolled in the clinical study. Patients were randomly assigned either 7.5 mg/kg nitazoxanide as an oral suspension or placebo twice a day for 3 days, and all remained in hospital for 7 days after start of treatment. The primary endpoint was time from first dose to resolution of illness, and analysis was by modified intention-to-treat. This study is registered with ClinicalTrials.gov, number NCT00302640. FINDINGS Survival analysis showed that the median time to resolution of illness was 31 h (IQR 22-73) for the nitazoxanide-treated group compared with 75 h (51-124) for the placebo group (p=0.0137). No significant adverse events were reported. INTERPRETATION A 3-day course of nitazoxanide significantly reduced the duration of rotavirus disease in hospitalised paediatric patients. These results are encouraging, and might lead us to think about new approaches to managing rotavirus disease in children.

Targeting the Heat Shock Factor 1 by RNA Interference: A Potent Tool to Enhance Hyperthermochemotherapy Efficacy in Cervical Cancer

Carcinoma of the uterine cervix is one of the highest causes of mortality in female cancer patients worldwide, and improved treatment options for this type of malignancy are highly needed. Local hyperthermia has been successfully used in combination with systemic administration of cisplatin-based chemotherapy in phase I/II clinical studies. Heat-induced expression of cytoprotective and antiapoptotic heat shock proteins (HSP) is a known complication of hyperthermia, resulting in thermotolerance and chemoresistance and hindering the efficacy of the combination therapy. Heat shock transcription factor 1 (HSF1) is the master regulator of heat-induced HSP expression. In the present report, we used small interfering RNA (siRNA) to silence HSF1 and to examine the effect of HSF1 loss of function on the response to hyperthermia and cisplatin-based chemotherapy in HeLa cervical carcinoma. We have identified the 322-nucleotide to 340-nucleotide HSF1 sequence as an ideal target for siRNA-mediated HSF1 silencing, have created a pSUPER-HSF1 vector able to potently suppress the HSF1 gene, and have generated for the first time human cancer cell lines with stable loss of HSF1 function. We report that, although it surprisingly does not affect cancer cell sensitivity to cisplatin or elevated temperatures up to 43 degrees C when administered separately, loss of HSF1 function causes a dramatic increase in sensitivity to hyperthermochemotherapy, leading to massive (>95%) apoptosis of cancer cells. These findings indicate that disruption of HSF1-induced cytoprotection during hyperthermochemotherapy may represent a powerful strategy to selectively amplify the damage in cancer cells and identify HSF1 as a promising therapeutic target in cervical carcinoma.

Herpes simplex virus disrupts NF-κB regulation by blocking its recruitment on the IκBα promoter and directing the factor on viral genes

Herpes simplex viruses (HSVs) are able to hijack the host-cell IκB kinase (IKK)/NF-κB pathway, which regulates critical cell functions from apoptosis to inflammatory responses; however, the molecular mechanisms involved and the outcome of the signaling dysregulation on the host-virus interaction are mostly unknown. Here we show that in human keratinocytes HSV-1 attains a sophisticated control of the IKK/NF-κB pathway, inducing two distinct temporally controlled waves of IKK activity and disrupting the NF-κB autoregulatory mechanism. Using chromatin immunoprecipitation we demonstrate that dysregulation of the NF-κB-response is mediated by a virus-induced block of NF-κB recruitment to the promoter of the IκBα gene, encoding the main NF-κB-inhibitor. We also show that HSV-1 redirects NF-κB recruitment to the promoter of ICP0, an immediate-early viral gene with a key role in promoting virus replication. The results reveal a new level of control of cellular functions by invading viruses and suggest that persistent NF-κB activation in HSV-1-infected cells, rather than being a host response to the virus, may play a positive role in promoting efficient viral replication.

Regulation of Cyclooxygenase-2 Expression by Heat: A Novel Aspect of Heat Shock Factor 1 Function in Human Cells

The heat-shock response, a fundamental defense mechanism against proteotoxic stress, is regulated by a family of heat-shock transcription factors (HSF). In humans HSF1 is considered the central regulator of heat-induced transcriptional responses. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat-shock genes encoding cytoprotective heat-shock proteins (HSP) with chaperone function. In addition to its cytoprotective function, HSF1 was recently hypothesized to play a more complex role, regulating the expression of non-HSP genes; however, the non-canonical role of HSF1 is still poorly understood. Herein we report that heat-stress promotes the expression of cyclooxygenase-2 (COX-2), a key regulator of inflammation controlling prostanoid and thromboxane synthesis, resulting in the production of high levels of prostaglandin-E2 in human cells. We show that heat-induced COX-2 expression is regulated at the transcriptional level via HSF1-mediated signaling and identify, by in-vitro reporter gene activity assay and deletion-mutant constructs analysis, the COX-2 heat-responsive promoter region and a new distal cis-acting HSE located at position −2495 from the transcription start site. As shown by ChIP analysis, HSF1 is recruited to the COX-2 promoter rapidly after heat treatment; by using shRNA-mediated HSF1 suppression and HSE-deletion from the COX-2 promoter, we demonstrate that HSF1 plays a central role in the transcriptional control of COX-2 by heat. Finally, COX-2 transcription is also induced at febrile temperatures in endothelial cells, suggesting that HSF1-dependent COX-2 expression could contribute to increasing blood prostaglandin levels during fever. The results identify COX-2 as a human non-classical heat-responsive gene, unveiling a new aspect of HSF1 function.

NEW EMBO MEMBER’S REVIEW: NF-κB and virus infection: who controls whom

Among the different definitions of viruses, ‘pirates of the cell’ is one of the most picturesque, but also one of the most appropriate. Viruses have been known for a long time to utilize a variety of strategies to penetrate cells and, once inside, to take over the host nucleic acid and protein synthesis machinery to build up their own components and produce large amounts of viral progeny. As their genomes carry a minimal amount of information, encoding only a few structural and regulatory proteins, viruses are largely dependent on their hosts for survival; however, despite their apparent simplicity, viruses have evolved different replicative strategies that are regulated in a sophisticated manner. During the last years, the study of the elaborate relationship between viruses and their hosts has led to the understanding of how viral pathogens not only are able to alter the host metabolism via their signaling proteins, but are also able to hijack cellular signaling pathways and transcription factors, and control them to their own advantage. In particular, the nuclear factor‐κB (NF‐κB) pathway appears to be an attractive target for common human viral pathogens. This review summarizes what is known about the control of NF‐κB by viruses, and discusses the possible outcome of NF‐κB activation during viral infection, which may benefit either the host or the pathogen.

Synergistic Effect of Nitazoxanide with Neuraminidase Inhibitors against Influenza A Viruses In Vitro

ABSTRACT The emergence of drug-resistant influenza A virus (IAV) strains represents a serious threat to global human health and underscores the need for novel approaches to anti-influenza chemotherapy. Combination therapy with drugs affecting different IAV targets represents an attractive option for influenza treatment. We have previously shown that the thiazolide anti-infective nitazoxanide (NTZ) inhibits H1N1 IAV replication by selectively blocking viral hemagglutinin maturation. Herein we investigate the anti-influenza activity of NTZ against a wide range of human and avian IAVs (H1N1, H3N2, H5N9, H7N1), including amantadine-resistant and oseltamivir-resistant strains, in vitro. We also investigate whether therapy with NTZ in combination with the neuraminidase inhibitors oseltamivir and zanamivir exerts synergistic, additive, or antagonistic antiviral effects against influenza viruses. NTZ was effective against all IAVs tested, with 50% inhibitory concentrations (IC50s) ranging from 0.9 to 3.2 μM, and selectivity indexes (SIs) ranging from >50 to >160, depending on the strain and the multiplicity of infection (MOI). Combination therapy studies were performed in cell culture-based assays using A/Puerto Rico/8/1934 (H1N1), A/WSN/1933 (H1N1), or avian A/chicken/Italy/9097/1997 (H5N9) IAVs; dose-effect analysis and synergism/antagonism quantification were performed using isobologram analysis according to the Chou-Talalay method. Combination index (CI) analysis indicated that NTZ and oseltamivir combination treatment was synergistic against A/Puerto Rico/8/1934 (H1N1) and A/WSN/1933 (H1N1) IAVs, with CI values ranging between 0.39 and 0.63, independently of the MOI used. Similar results were obtained when NTZ was administered in combination with zanamivir (CI = 0.3 to 0.48). NTZ-oseltamivir combination treatment was synergistic also against the avian A/chicken/Italy/9097/1997 (H5N9) IAV (CI = 0.18 to 0.31). Taken together, the results suggest that regimens that combine neuraminidase inhibitors and nitazoxanide exert synergistic anti-influenza effects.

Ferruccio Ritossa’s scientific legacy 50 years after his discovery of the heat shock response: a new view of biology, a new society, and a new journal

The pioneering discovery of the heat shock response by the Italian scientist Ferruccio Ritossa reached maturity this year, 2012. It was 50 years ago that Professor Ritossa, through an extraordinary combination of serendipity, curiosity, knowledge and inspiration, published the first observation that cells could mount very strong transcriptional activity when exposed to elevated temperatures, which was coined the heat shock response. This discovery led to the identification of heat shock proteins, which impact many areas of current biology and medicine, and has created a new avenue for more exciting discoveries. In recognition of the discovery of the heat shock response, Cell Stress Society International (CSSI) awarded Professor Ritossa with the CSSI medallion in October 2010 in Dozza, Italy. This article is based on a session of the Fifth CSSI Congress held in Québec commemorating Professor Ritossa and his discovery.