Investigation of the Presence of DNA in Human Blood Plasma Small Extracellular Vesicles.

Extracellular DNA (ecDNA) is DNA outside of cells, which is a result of various mechanisms. EcDNA is believed to be a cause of various pathogeneses as well as their potential biomarker. EcDNA is believed to also be part of small extracellular vesicles (sEVs) from cell cultures. If ecDNA is present in sEVs in plasma, their membrane may protect it from degradation by deoxyribonucleases. Moreover, sEVs play a role in the intercellular communication, and they can therefore transfer ecDNA between cells. The aim of this study was to investigate the presence of ecDNA in sEVs isolated from fresh human plasma by the ultracentrifugation and density gradient, which serves to exclude the co-isolation of non-sEVs compartments. The novelty of the current study is the investigation of the localization and subcellular origin of the ecDNA associated with sEVs in plasma, as well as the estimation of the approximate concentration. The cup-shaped sEVs were confirmed by transmission electron microscopy. The highest concentration of particles was in the size of 123 nm. The presence of the sEVs markers CD9 and TSG101 was confirmed by western blot. It was found that 60-75% of DNA is on the surface of sEVs, but a part of the DNA is localized inside the sEVs. Moreover, both nuclear and mitochondrial DNA were present in plasma EVs. Further studies should focus on the potential harmful autoimmune effect of DNA carried by plasma EVs or specifically sEVs.

Regulation of AGR2 expression via 3'UTR shortening.

One recently discussed general mechanism affecting gene expression is 3'-untranslated region (3'UTR) length. Events such as shortening, translocation or loss of 3'UTRs are observed within oncogenes and are proposed to associate with increased expression. Thus, increased efforts are being made to understand constitutive and differential transcript 3'end formation. Investigation of AGR2 mRNA revealed a direct impact of its 3'UTR length on AGR2 expression. In silico analyses identified several regulatory sequences within the distal part of AGR2 mRNA that may regulate 3'UTR length and associated protein levels. Short 3'UTRs were observed in a panel of AGR2-positive cancer cell lines and in human breast cancer specimens, in which more extensive 3'UTR shortening correlated with increased AGR2 protein levels. AGR2 is an important member of PI3K/AKT signalling pathway, which along with the proposed involvement of mTOR in the regulation of alternative polyadenylation, prompted us to study the role of mTOR in relation to AGR2 mRNA 3'UTR shortening. A direct impact of mTOR signalling on AGR2 3'UTR shortening associated with increased protein synthesis was found, which led to the identification of a novel molecular mechanism involved in upregulation of AGR2 levels in mTOR-activated cells via modulating the 3'UTR length of AGR2 mRNA.

Apoptosis-induced ectodomain shedding of hypoxia-regulated carbonic anhydrase IX from tumor cells: a double-edged response to chemotherapy.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin. METHODS: Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay. RESULTS: We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness. CONCLUSIONS: These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.

Enriching the drinking water of rats with extracts of Salvia officinalis and Thymus vulgaris increases their resistance to oxidative stress.

Nature is an attractive source of therapeutic compounds. In comparison to the artificial drugs, natural compounds cause less adverse side effects and are suitable for current molecularly oriented approaches to drug development and their mutual combining. Medicinal plants represent one of the most available remedy against various diseases. Proper examples are Salvia officinalis L. and Thymus vulgaris L. which are known aromatic medicinal plants. They are very popular and frequently used in many countries. The molecular mechanism of their biological activity has not yet been fully understood. The aim of this study was to ascertain if liver cells of experimental animals drinking extracts of sage or thyme will manifest increased resistance against oxidative stress. Adult Sprague-Dawley rats were divided into seven groups. They drank sage or thyme extracts for 2 weeks. At the end of the drinking period, blood samples were collected for determination of liver biochemical parameters and hepatocytes were isolated to analyze (i) oxidatively generated DNA damage (conventional and modified comet assay), (ii) activities of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx)] and (iii) content of glutathione. Intake of sage and thyme had no effect either on the basal level of DNA damage or on the activity of SOD in rat hepatocytes and did not change the biochemical parameters of blood plasma. Simultaneously, the activity of GPx was significantly increased and the level of DNA damage induced by oxidants was decreased. Moreover, sage extract was able to start up the antioxidant protection expressed by increased content of glutathione. Our results indicate that the consumption of S.officinalis and T.vulgaris extracts positively affects resistency of rat liver cells against oxidative stress and may have hepatoprotective potential.

Extracellular DNA and Markers of Neutrophil Extracellular Traps in Saliva from Patients with Periodontitis-A Case-Control Study.

Periodontitis is a chronic inflammatory disease. We have previously shown that salivary DNA is higher in patients with periodontitis. Neutrophil extracellular traps (NETs) are involved in the pathogenesis of chronic inflammatory diseases. The objective of this case-control study was to compare patients with periodontitis and healthy controls regarding the salivary concentrations of extracellular DNA and NET components. Unstimulated saliva samples were collected from 49 patients with periodontitis and 71 controls before an oral examination. Salivary extracellular DNA was isolated and quantified fluorometrically and using PCR. NET-associated markers were assessed using ELISA. We have found significantly higher concentrations of salivary extracellular DNA in samples from periodontitis patients (five-times higher for supernatant and three times for pellet). Our results show that patients also have three-times-higher salivary nucleosomes and NET-associated enzymes-myeloperoxidase and neutrophil elastase (both two-times higher). Neutrophil elastase and salivary DNA in the pellet correlated positively with the pocket depth/clinical attachment level in periodontitis patients (r = 0.31-weak correlation; p = 0.03 and r = 0.41-moderate correlation, p = 0.004). Correlations between salivary extracellular DNA and NET enzymes were positive and significant. Based on our results, the higher salivary extracellular DNA in periodontitis seems to be related to components of NETs, albeit with weak to moderate correlations indicating that NETs are produced in periodontitis and can play a role in its pathogenesis similarly to other inflammatory diseases. Further studies should prove this assumption with potential diagnostic and therapeutic consequences.

Effects of exogenous deoxyribonuclease I in collagen antibody-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is associated with a high concentration of extracellular DNA (ecDNA). This could be a consequence of the inflammation, but the ecDNA could also be involved in the unknown etiopathogenesis of RA. Clearance of ecDNA is hypothesized to prevent the development of RA. This study aimed to analyze the effects of exogenous deoxyribonuclease I (DNase I) administration in an animal model of RA. METHODS: The collagen antibody-induced arthritis (CAIA) model of RA was induced in adult female DBA/1J mice. CAIA mice were treated with saline or DNase I (10 mg/kg) every 12 h for the whole duration of the experiment. Arthritic scores were assessed. Paw volume and temperature were assessed using a plethysmometer and a thermal camera, respectively. Plasma ecDNA and its subcellular origin were analyzed using fluorometry and real-time PCR. DNase activity was quantified with single radial enzyme diffusion method. RESULTS: The CAIA model was successfully induced as proved by a higher volume, temperature and the overall arthritis score in comparison to controls. The administration of DNase I resulted in a nearly two-fold increase in serum DNase activity. Still, it did affect neither plasma ecDNA, nor the arthritis score or other measures of joint inflammation. CONCLUSION: Our results suggest that exogenous DNase I does not prevent the development of CAIA in mice. Whether this is true for other animal models of arthritis or clinical RA requires further research. EcDNA does not seem to be involved in the pathogenesis of CAIA. Additional studies are also needed to elucidate the role of ecDNA in the development of RA, focusing especially on its origin and inhibition of ecDNA release.

Mitochondrial DNA variability and Covid-19 in the Slovak population.

Recent studies have shown that mitochondria are involved in the pathogenesis of Covid-19. Mitochondria play a role in production of reactive oxygen species and induction of an innate immune response, both important during infections. Common variability of mitochondrial DNA (mtDNA) can affect oxidative phosphorylation and the risk or lethality of cardiovascular, neurodegenerative diseases and sepsis. However, it is unclear whether susceptibility of severe Covid-19 might be affected by mtDNA variation. Thus, we have analyzed mtDNA in a sample of 446 Slovak patients hospitalized due to Covid-19 and a control population group consisting of 1874 individuals. MtDNA variants in the HVRI region have been analyzed and classified into haplogroups at various phylogenetic levels. Binary logistic regression was used to assess the risk of Covid-19. Haplogroups T1, H11, K and variants 16256C > T, 16265A > C, 16293A > G, 16311 T > C and 16399A > G were associated with an increased Covid-19 risk. On contrary, Haplogroup J1, haplogroup clusters H + U5b and T2b + U5b, and the mtDNA variant 16189 T > C were associated with decreased risk of Covid-19. Following the application of the Bonferroni correction, statistical significance was observed exclusively for the cluster of haplogroups H + U5b. Unsurprisingly, the most significant factor contributing to the mortality of patients with Covid-19 is the age of patients. Our findings suggest that mtDNA haplogroups can play a role in Covid-19 pathogenesis, thus potentially useful in identifying susceptibility to its severe form. To confirm these associations, further studies taking into account the nuclear genome or other non-biological influences are needed.

Neutrophil extracellular traps formation and clearance is enhanced in fever and attenuated in hypothermia.

Fever and hypothermia represent two opposite strategies for fighting systemic inflammation. Fever results in immune activation; hypothermia is associated with energy conservation. Systemic Inflammatory Response Syndrome (SIRS) remains a significant cause of mortality worldwide. SIRS can lead to a broad spectrum of clinical symptoms but importantly, patients can develop fever or hypothermia. During infection, polymorphonuclear cells (PMNs) such as neutrophils prevent pathogen dissemination through the formation of neutrophil extracellular traps (NETs) that ensnare and kill bacteria. However, when dysregulated, NETs also promote host tissue damage. Herein, we tested the hypothesis that temperature modulates NETs homeostasis in response to infection and inflammation. NETs formation was studied in response to infectious (Escherichia coli, Staphylococcus aureus) and sterile (mitochondria) agents. When compared to body temperature (37°C), NETs formation increased at 40°C; interestingly, the response was stunted at 35°C and 42°C. While CD16+ CD49d+ PMNs represent a small proportion of the neutrophil population, they formed ~45-85% of NETs irrespective of temperature. Temperature increased formyl peptide receptor 1 (FPR1) expression to a differential extent in CD16+ CD49d- vs. CD49d+ PMNSs, suggesting further complexity to neutrophil function in hypo/hyperthermic conditions. The capacity of NETs to induce Toll-like receptor 9 (TLR9)-mediated NF-κB activation was found to be temperature independent. Interestingly, NET degradation was enhanced at higher temperatures, which corresponded with greater plasma DNase activity in response to temperature increase. Collectively, our observations indicate that NETs formation and clearance are enhanced at 40°C whilst temperatures of 35°C and 42°C attenuate this response. Targeting PMN-driven immunity may represent new venues for intervention in pathological inflammation.

Severe gestational diabetes mellitus in lean dams is associated with low IL-1α levels and affects the growth of the juvenile mouse offspring.

We investigated how maternal gestational diabetes (GDM) impacts the metabolic status of offspring. GDM was induced in CD1 mice consuming a fast-food diet (FFD) by repeated low-dose streptozotocin injections before mating. Offspring of normoglycemic standard chow or the FFD consuming dams served as controls. In 4-week-old offspring weaned to standard chow, plasma concentrations of extracellular DNA, inflammatory markers, and parameters of the cardiometabolic status (glycemia, liver lipid content; body, organ, and fat weight) were determined. Two-factor analysis of variance indicated that the male offspring of GDM dams manifest postnatal growth retardation and lower relative kidney weight. Regardless of sex, GDM offspring manifest the lowest IL-1α levels, and other inflammatory markers showed mild and inconsistent alterations. Offspring of dams consuming the FFD displayed higher liver triacylglycerols content. The three groups of offspring showed no significant differences in glycemia and extracellular DNA. Partial least squares-discriminant analysis indicated that male GDM offspring present lower kidney, body, and brown adipose tissue weights; lower IL-1α levels, and higher concentrations of GM-CSF and IL-10 compared with their FFD counterparts. The model failed to select discriminative variables in females. In conclusion, in mice, maternal GDM in the absence of obesity adversely affects the early growth of juvenile male offspring.

Neutrophil extracellular traps in urinary tract infection.

Background: Urinary tract infections (UTI) are common types of bacterial infection in children. UTI treatment is aimed to prevent complications including hypertension, proteinuria, and progression to chronic kidney disease. Activated neutrophils release chromatin-based structures associated with antimicrobial proteins called neutrophil extracellular traps (NETs). We aimed to describe the role of NET-associated markers in children with UTI as well as the role of NETs formation in a mouse model of UTI. Materials and methods: Markers of NETs including extracellular DNA (ecDNA), myeloperoxidase (MPO) and cathelicidin were analyzed in children with febrile UTI caused by E. coli (n = 98, aged 0.3-1.3 years) and in healthy controls (n = 50, 0.5-5.2 years). Moreover, an acute experimental model of UTI was performed on PAD4 knock-out mice with diminished NETs formation (n = 18), and on wild-type mice (n = 15). Results: Children with UTI had significantly higher urinary NETs markers including total ecDNA, nuclear DNA and mitochondrial DNA, altogether with MPO and cathelicidin. The concentrations of MPO and cathelicidin positively correlated with ecDNA (r = 0.53, p ≤ 0.001; r = 0.56, p ≤ 0.001, respectively) and the number of leukocytes in the urine (r = 0.29, p ≤ 0.05; r = 0.27, p ≤ 0.05, respectively). Moreover, urinary MPO was positively associated with cathelicidin (r = 0.61, p ≤ 0.001). In the experimental model, bacterial load in the bladder (20-fold) and kidneys (300-fold) was significantly higher in PAD4 knock-out mice than in wild-type mice. Conclusion: Higher urinary NETs makers-ecDNA, MPO and cathelicidin and their correlation with leukocyturia in children with UTI confirmed our hypothesis about the association between NETs and UTI in children. Higher bacterial load in mice with diminished NETs formation suggests that NETs are not only a simple consequence of UTI, but might play a direct role in the prevention of pyelonephritis and other UTI complications.

Mitochondria-induced formation of neutrophil extracellular traps is enhanced in the elderly via Toll-like receptor 9.

Neutrophil extracellular traps are potent antimicrobial weapons; however, their formation during sterile inflammation is detrimental, and the mechanism of induction is still unclear. Since advanced age is the primary clinical risk factor for poor outcomes in inflammatory diseases, we hypothesized that sterile stimuli, represented by mitochondria, would induce neutrophil extracellular trap formation in an age-dependent manner. Therefore, we analyzed induction of neutrophil extracellular traps in patients grouped according to age or immune status and observed that neutrophils from elderly patients responded to the presence of mitochondria with enhanced neutrophil extracellular trap formation. These neutrophil extracellular traps were also found to be more oxidized and exhibited higher resistance to DNase I degradation. Additionally, a higher concentration of residual neutrophil extracellular traps was detected in the plasma of the elderly. This plasma was capable of priming neutrophils through TLR9-mediated signaling, leading to further neutrophil extracellular trap formation, which was successfully inhibited with chloroquine. Finally, in a mouse model of mitochondria-induced acute lung injury, we observed that neutrophils from aged mice displayed impaired chemotactic activity but exhibited a trend of higher neutrophil extracellular trap formation. Thus, we propose that residual neutrophil extracellular traps circulating in the elderly preactivate neutrophils, making them more prone to enhanced neutrophil extracellular trap formation when exposed to mitochondria during sterile inflammation. Further investigation is needed to determine whether this vicious circle could be a suitable therapeutic target.

Sex differences in long-term effects of collagen-induced arthritis in middle-aged mice.

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disorder with high prevalence among middle-aged women. Collagen-induced arthritis (CIA) is the most widely used animal model of RA, however, sex differences and long-term effects of CIA in mice are poorly described in the literature. Aim: Therefore, the present study aimed to analyze the long-term effects of CIA on the joints of middle-aged mice of both sexes and to describe potential sex differences. Materials and methods: CIA was induced in middle-aged DBA/1J mice by immunization with bovine type II collagen and complete Freund's adjuvant. Saline was administered to control mice. Arthritis score assessment, plethysmometry, and thermal imaging of the joints were performed weekly for 15 weeks. Locomotor activity, micro-computed tomography, joint histology and biochemical analyses were performed at the end of the experiment. Results: Our results indicate a similar prevalence of arthritis in both sexes of mice-67% (8/12) of females and 89% (8/9) males with an earlier onset in males (day 14 vs. day 35). After the arthritis scores peaked on day 56 for males and day 63 for females, they steadily declined until the end of the experiment on day 105. A similar dynamics was observed in paw volume and temperature analyzing different aspects of joint inflammation. Long-term consequences including higher proteinuria (by 116%), loss of bone density (by 33.5%) and joint damage in terms of synovial hyperplasia as well as bone and cartilage erosions were more severe in CIA males compared to CIA females. There were no significant differences in locomotor activity between CIA mice and CTRL mice of any sex. Conclusion: This is the first study to describe the long-term effects of the CIA model in terms of sex differences in DBA/1J mice. Our results indicate sex differences in the dynamics, but not in the extent of arthritis. An earlier onset of arthritis and more severe consequences on joints, bones and kidneys were found in males. The underlying immune pathomechanisms responsible for the limited duration of the arthritis symptoms and the opposite sex difference in comparison to RA patients require further investigation.

Cognitive impairment and biomarkers of gut microbial translocation in testicular germ cell tumor survivors.

Background: Survivors of testicular germ cell tumors (GCT) may suffer from late cognitive impairment. We hypothesized that disruption of intestinal barrier during chemotherapy and/or radiotherapy may be a contributing factor of cognitive dysfunction within the gut-blood-brain axis. Methods: GCT survivors (N = 142) from National Cancer Institute of Slovakia completed the Functional Assessment of Cancer Therapy Cognitive Function questionnaires during their annual follow-up visit at 9-year median (range 4-32). Biomarkers of gut microbial translocation and dysbiosis high mobility group box-1 (HMGB-1), lipopolysaccharide, d-lactate and sCD14 were measured from peripheral blood obtained during the same visit. Each questionnaire score was correlated with biomarkers. Survivors were treated with orchiectomy only (N = 17), cisplatin-based chemotherapy (N = 108), radiotherapy to the retroperitoneum (N = 11) or both (N = 6). Results: GCT survivors with higher sCD14 (above median) had worse cognitive function perceived by others (CogOth domain) (mean ± SEM; 14.6 ± 0.25 vs 15.4 ± 0.25, p = 0.019), lower perceived cognitive abilities (CogPCA domain) (20.0 ± 0.74 vs 23.4 ± 0.73, p = 0.025) and lower overall cognitive function score (109.2 ± 0.74 vs 116.7 ± 1.90, p = 0.021). There were no significant cognitive declines associated with HMGB-1, d-lactate and lipopolysaccharide. Survivors treated with ≥ 400mg/m2 vs < 400mg/m2 of cisplatin-based chemotherapy had a higher lipopolysaccharide (567.8 µg/L ± 42.7 vs 462.9 µg/L ± 51.9, (p = 0.03). Conclusions: sCD14 is a marker of monocytic activation by lipopolysaccharide and may also serve as a promising biomarker of cognitive impairment in long-term cancer survivors. While chemotherapy and radiotherapy-induced intestinal injury may be the underlying mechanism, further research using animal models and larger patient cohorts are needed to explore the pathogenesis of cognitive impairment in GCT survivors within the gut-brain axis.

On the Origin of Neutrophil Extracellular Traps in COVID-19.

Despite ongoing vaccination COVID-19 is a global healthcare problem because of the lack of an effective targeted therapy. In severe COVID-19 manifesting as acute respiratory distress syndrome, uncontrolled innate immune system activation results in cytokine deregulation, damage-associated molecular patterns release upon tissue damage and high occurrence of thrombotic events. These pathomechanisms are linked to neutrophil function and dysfunction, particularly increased formation of neutrophil extracellular traps (NETs). While the association of NETs and severity of COVID-19 has been shown and proved, the causes of NETs formation are unclear. The aim of this review is to summarize potential inducers of NETs formation in severe COVID-19 and to discuss potential treatment options targeting NETs formation of removal.

Molecular actions of sex hormones in the brain and their potential treatment use in anxiety disorders.

Anxiety disorders are one of the most prevalent mood disorders that can lead to impaired quality of life. Current treatment of anxiety disorders has various adverse effects, safety concerns, or restricted efficacy; therefore, novel therapeutic targets need to be studied. Sex steroid hormones (SSHs) play a crucial role in the formation of brain structures, including regions of the limbic system and prefrontal cortex during perinatal development. In the brain, SSHs have activational and organizational effects mediated by either intracellular or transmembrane G-protein coupled receptors. During perinatal developmental periods, the physiological concentrations of SSHs lead to the normal development of the brain; however, the early hormonal dysregulation could result in various anxiety diorders later in life. Sex differences in the prevalence of anxiety disorders suggest that SSHs might be implicated in their development. In this review, we discuss preclinical and clinical studies regarding the role of dysregulated SSHs signaling during early brain development that modifies the risk for anxiety disorders in a sex-specific manner in adulthood. Moreover, our aim is to summarize potential molecular mechanisms by which the SSHs may affect anxiety disorders in preclinical research. Finally, the potential effects of SSHs in the treatment of anxiety disorders are discussed.

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

The Role of HSF1 Protein in Malignant Transformation.

BACKGROUND: The heat shock transcription factor, HSF1, is the main regulator of the proteotoxic stress response that orchestrates the adaptation of cells to stress conditions such as elevated temperature, oxidative stress, and proteotoxic stress. As such, HSF1 regulates a large number of stress response-related genes, primarily those encoding heat shock proteins (HSPs). HSPs are molecular chaperones involved in the acquisition of native protein conformations and the prevention of protein degradation, and they also contribute to the removal of denatured proteins via the proteasome. Representative members of the HSP family are HSP70 and HSP90. The stress response is a highly conserved mechanism across all eukaryotes, and HSF1 has been linked to a number of physiological processes (ribosomal biogenesis, translation, transcription, cell cycle, and metabolism) and pathological disorders (neurodegenerative disorders such as Parkinson´s and Alzheimer´s diseases). HSF1 activation is also prominent in different types of cancer (prostate, breast, colorectal carcinoma etc.) where it correlates with tumor aggressiveness and poor prognosis. HSF1 is therefore considered a diagnostic and prognostic marker and is currently being targeted to develop new cancer therapies. Several inhibitors of HSF1 have already been synthesized, but their molecular mechanism (s) of action, specificity those of HSF1, nontoxicity in healthy tissues, and their efficacy in targeting tumor cells remain to be elucidated. PURPOSE: This review summarizes known mechanisms of HSF1 regulation and activation, the role of HSF1 during malignant transformation, and the potential of designing small molecule HSF1 inhibitors for cancer therapy. Key words: HSF1 transcription factor - molecular chaperones - cellular stress - tumor transformation - cancer This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 10. 8. 2018.

Cancer-associated S100P protein binds and inactivates p53, permits therapy-induced senescence and supports chemoresistance.

S100P belongs to the S100 family of calcium-binding proteins regulating diverse cellular processes. Certain S100 family members (S100A4 and S100B) are associated with cancer and used as biomarkers of metastatic phenotype. Also S100P is abnormally expressed in tumors and implicated in migration-invasion, survival, and response to therapy. Here we show that S100P binds the tumor suppressor protein p53 as well as its negative regulator HDM2, and that this interaction perturbs the p53-HDM2 binding and increases the p53 level. Paradoxically, the S100P-induced p53 is unable to activate its transcriptional targets hdm2, p21WAF, and bax following the DNA damage. This appears to be related to reduced phosphorylation of serine residues in both N-terminal and C-terminal regions of the p53 molecule. Furthermore, the S100P expression results in lower levels of pro-apoptotic proteins, in reduced cell death response to cytotoxic treatments, followed by stimulation of therapy-induced senescence and increased clonogenic survival. Conversely, the S100P silencing suppresses the ability of cancer cells to survive the DNA damage and form colonies. Thus, we propose that the oncogenic role of S100P involves binding and inactivation of p53, which leads to aberrant DNA damage responses linked with senescence and escape to proliferation. Thereby, the S100P protein may contribute to the outgrowth of aggressive tumor cells resistant to cytotoxic therapy and promote cancer progression.

Sulforaphane-induced apoptosis involves the type 1 IP3 receptor.

In this study we show that anti-tumor effect of sulforaphane (SFN) is partially realized through the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). This effect was verified in vitro on three different stable cell lines and also in vivo on the model of nude mice with developed tumors. Early response (6 hours) of A2780 ovarian carcinoma cells to SFN treatment involves generation of mitochondrial ROS and increased transcription of NRF2 and its downstream regulated genes including heme oxygenase 1, NAD(P)H:quinine oxidoreductase 1, and KLF9. Prolonged SFN treatment (24 hours) upregulated expression of NRF2 and IP3R1. SFN induces a time-dependent phosphorylation wave of HSP27. Use of IP3R inhibitor Xestospongin C (Xest) attenuates both SFN-induced apoptosis and the level of NRF2 protein expression. In addition, Xest partially attenuates anti-tumor effect of SFN in vivo. SFN-induced apoptosis is completely inhibited by silencing of IP3R1 gene but only partially blocked by silencing of NRF2; silencing of IP3R2 and IP3R3 had no effect on these cells. Xest inhibitor does not significantly modify SFN-induced increase in the rapid activity of ARE and AP1 responsive elements. We found that Xest effectively reverses the SFN-dependent increase of nuclear content and decrease of reticular calcium content. In addition, immunofluorescent staining with IP3R1 antibody revealed that SFN treatment induces translocation of IP3R1 to the nucleus. Our results clearly show that IP3R1 is involved in SFN-induced apoptosis through the depletion of reticular calcium and modulation of transcription factors through nuclear calcium up-regulation.

Sulforaphane reduces molecular response to hypoxia in ovarian tumor cells independently of their resistance to chemotherapy.

One of the recently emerging anticancer strategies is the use of natural dietary compounds, such as sulforaphane, a cancer-chemopreventive isothiocyanate found in broccoli. Based on the growing evidence, sulforaphane acts through molecular mechanisms that interfere with multiple oncogenic pathways in diverse tumor cell types. Herein, we investigated the anticancer effects of bioavailable concentrations of sulforaphane in ovarian carcinoma cell line A2780 and its two derivatives, adriamycin-resistant A2780/ADR and cisplatin-resistant A2780/CP cell lines. Since tumor microenvironment is characterized by reduced oxygenation that induces aggressive tumor phenotype (such as increased invasiveness and resistance to chemotherapy), we evaluated the effects of sulforaphane in ovarian cancer cells exposed to hypoxia (2% O2). Using the cell-based reporter assay, we identified several oncogenic pathways modulated by sulforaphane in hypoxia by activating anticancer responses (p53, ARE, IRF-1, Pax-6 and XRE) and suppressing responses supporting tumor progression (AP-1 and HIF-1). We further showed that sulforaphane decreases the level of HIF-1α protein without affecting its transcription and stability. It can also diminish transcription and protein level of the HIF-1 target, CA IX, which protects tumor cells from hypoxia-induced pH imbalance and facilitates their migration/invasion. Accordingly, sulforaphane treatment leads to diminished pH regulation and reduced migration of ovarian carcinoma cells. These effects occur in all three ovarian cell lines suggesting that sulforaphane can overcome the chemoresistance of cancer cells. This offers a path potentially exploitable in sensitizing resistant cancer cells to therapy, and opens a window for the combined treatments of sulforaphane either with conventional chemotherapy, natural compounds, or with other small molecules.